Artificial antibody polypeptides

ABSTRACT

The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

Portions of the present invention were made with support of the UnitedStates Government via a grant from the National Institutes of Healthunder grant number GM 55042 The U.S. Government therefore may havecertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of the productionand selection of binding and catalytic polypeptides by the methods ofmolecular biology. The invention specifically relates to the generationof both nucleic acid and polypeptide libraries encoding the molecularscaffolding of a modified Fibronectin Type III (Fn3) molecule. Theinvention also relates to “artificial mini-antibodies” or “monobodies,”i.e., polypeptides containing an Fn3 scaffold onto which loop regionscapable of binding to a variety of different molecular structures (suchas antibody binding sites) have been grafted.

BACKGROUND OF THE INVENTION Antibody Structure

A standard antibody (Ab) is a tetrameric structure consisting of twoidentical immunoglobulin (Ig) heavy chains and two identical lightchains. The heavy and light chains of an Ab consist of differentdomains. Each light chain has one variable domain (VL) and one constantdomain (CL), while each heavy chain has one variable domain (VH) andthree or four constant domains (CH) (Alzari et al., 1988). Each domain,consisting of ˜110 amino acid residues, is folded into a characteristicβ-sandwich structure formed from two β-sheets packed against each other,the immunoglobulin fold. The VH and VL domains each have threecomplementarity determining regions (CDR1-3) that are loops, or turns,connecting β-strands at one end of the domains (FIG. 1: A, C). Thevariable regions of both the light and heavy chains generally contributeto antigen specificity, although the contribution of the individualchains to specificity is not always equal. Antibody molecules haveevolved to bind to a large number of molecules by using six randomizedloops (CDRs). However, the size of the antibodies and the complexity ofsix loops represents a major design hurdle if the end result is to be arelatively small peptide ligand.

Antibody Substructures

Functional substructures of Abs can be prepared by proteolysis and byrecombinant methods. They include the Fab fragment, which contains theVH-CH1 domains of the heavy chain and the VL-CL1 domains of the lightchain joined by a single interchain disulfide bond, and the Fv fragment,which contains only the VH and VL domains. In some cases, a single VHdomain retains significant affinity (Ward et al., 1989). It has alsobeen shown that a certain monomeric κ light chain will specifically bindto its cognate antigen. (L. Masat et al., 1994). Separated light orheavy chains have sometimes been found to retain some antigen-bindingactivity (Ward et al., 1989). These antibody fragments are not suitablefor structural analysis using NMR spectroscopy due to their size, lowsolubility or low conformational stability.

Another functional substructure is a single chain Fv (scFv), made of thevariable regions of the immunoglobulin heavy and light chain, covalentlyconnected by a peptide linker (S-z Hu et al., 1996). These small (M_(r)25,000) proteins generally retain specificity and affinity for antigenin a single polypeptide and can provide a convenient building block forlarger, antigen-specific molecules. Several groups have reportedbiodistribution studies in xenografted athymic mice using scFv reactiveagainst a variety of tumor antigens, in which specific tumorlocalization has been observed. However, the short persistence of scFvsin the circulation limits the exposure of tumor cells to the scFvs,placing limits on the level of uptake. As a result, tumor uptake byscFvs in animal studies has generally been only 1-5% ID/g as opposed tointact antibodies that can localize in tumors ad 30-40% ID/g and havereached levels as high as 60-70% ID/g.

A small protein scaffold called a “minibody” was designed using a partof the Ig VH domain as the template (Pessi et al., 1993). Minibodieswith high affinity (dissociation constant (K_(d))˜10⁻⁷M) tointerleukin-6 were identified by randomizing loops corresponding to CDR1and CDR2 of VH and then selecting mutants using the phage display method(Martin et al., 1994). These experiments demonstrated that the essenceof the Ab function could be transferred to a smaller system. However,the minibody had inherited the limited solubility of the VH domain(Bianchi et al., 1994).

It has been reported that camels (Camelus dromedarius) often lackvariable light chain domains when IgG-like material from their serum isanalyzed, suggesting that sufficient antibody specificity and affinitycan be derived form VH domains (three CDR loops) alone. Davies andRiechmann recently demonstrated that “camelized” VH domains with highaffinity (K_(d)˜10⁻⁷ M) and high specificity can be generated byrandomizing only the CDR3. To improve the solubility and suppressnonspecific binding, three mutations were introduced to the frameworkregion (Davies & Riechmann, 1995). It has not been definitively shown,however, that camelization can be used, in general, to improve thesolubility and stability of VHs.

An alternative to the “minibody” is the “diabody.” Diabodies are smallbivalent and bispecific antibody fragments, i.e., they have twoantigen-binding sites. The fragments contain a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) on thesame polypeptide chain (V_(H)-V_(L)). Diabodies are similar in size toan Fab fragment. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or “diabodies,”are bivalent and bispecific (P. Holliger et al., 1993).

Since the development of the monoclonal antibody technology, a largenumber of 3D structures of Ab fragments in the complexed and/or freestates have been solved by X-ray crystallography (Webster et al., 1994;Wilson & Stanfield, 1994). Analysis of Ab structures has revealed thatfive out of the six CDRs have limited numbers of peptide backboneconformations, thereby permitting one to predict the backboneconformation of CDRs using the so-called canonical structures (Lesk &Tramontano, 1992; Rees et al., 1994). The analysis also has revealedthat the CDR3 of the VH domain (VH-CDR3) usually has the largest contactsurface and that its conformation is too diverse for canonicalstructures to be defined; VH-CDR3 is also known to have a largevariation in length (Wu et al., 1993). Therefore, the structures ofcrucial regions of the Ab-antigen interface still need to beexperimentally determined.

Comparison of crystal structures between the free and complexed stateshas revealed several types of conformational rearrangements. Theyinclude side-chain rearrangements, segmental movements, largerearrangements of VH-CDR3 and changes in the relative position of the VHand VL domains (Wilson & Stanfield, 1993). In the free state, CDRs, inparticular those which undergo large conformational changes uponbinding, are expected to be flexible. Since X-ray crystallography is notsuited for characterizing flexible parts of molecules, structuralstudies in the solution state have not been possible to provide dynamicpictures of the conformation of antigen-binding sites.

Mimicking the Antibody-Binding Site

CDR peptides and organic CDR mimetics have been made (Dougall et al.,1994). CDR peptides are short, typically cyclic, peptides whichcorrespond to the amino acid sequences of CDR loops of antibodies. CDRloops are responsible for antibody-antigen interactions. Organic CDRmimetics are peptides corresponding to CDR loops which are attached to ascaffold, e.g., a small organic compound.

CDR peptides and organic CDR mimetics have been shown to retain somebinding affinity (Smyth & von Itzstein, 1994). However, as expected,they are too small and too flexible to maintain full affinity andspecificity. Mouse CDRs have been grafted onto the human Ig frameworkwithout the loss of affinity (Jones et al., 1986; Riechmann et al.,1988), though this “humanization” does not solve the above-mentionedproblems specific to solution studies.

Mimicking Natural Selection Processes of Abs

In the immune system, specific Abs are selected and amplified from alarge library (affinity maturation). The processes can be reproduced invitro using combinatorial library technologies. The successful displayof Ab fragments on the surface of bacteriophage has made it possible togenerate and screen a vast number of CDR mutations (McCafferty et al.,1990; Barbas et al., 1991; Winter et al., 1994). An increasing number ofFabs and Fvs (and their derivatives) is produced by this technique,providing a rich source for structural studies. The combinatorialtechnique can be combined with Ab mimics.

A number of protein domains that could potentially serve as proteinscaffolds have been expressed as fusions with phage capsid proteins.Review in Clackson & Wells, Trends Biotechnol. 12:173-184 (1994).Indeed, several of these protein domains have already been used asscaffolds for displaying random peptide sequences, including bovinepancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433 (1992)),human growth hormone (Lowman et al., Biochemistry 30:10832-10838(1991)), Venturini et al., Protein Peptide Letters 1:70-75 (1994)), andthe IgG binding domain of Streptococcus (O'Neil et al., Techniques inProtein Chemistry V (Crabb, L., ed.) pp. 517-524, Academic Press, SanDiego (1994)). These scaffolds have displayed a single randomized loopor region.

Researchers have used the small 74 amino acid α-amylase inhibitorTendamistat as a presentation scaffold on the filamentous phage M13(McConnell and Hoess, 1995). Tendamistat is a β-sheet protein fromStreptomyces tendae. It has a number of features that make it anattractive scaffold for peptides, including its small size, stability,and the availability of high resolution NMR and X-ray structural data.Tendamistat's overall topology is similar to that of an immunoglobulindomain, with two β-sheets connected by a series of loops. In contrast toimmunoglobulin domains, the β-sheets of Tendamistat are held togetherwith two rather than one disulfide bond, accounting for the considerablestability of the protein. By analogy with the CDR loops found inimmunoglobulins, the loops the Tendamistat may serve a similar functionand can be easily randomized by in vitro mutagenesis.

Tendamistat, however, is derived from Streptomyces tendae. Thus, whileTendamistat may be antigenic in humans, its small size may reduce orinhibit its antigenicity. Also, Tendamistat's stability is uncertain.Further, the stability that is reported for Tendamistat is attributed tothe presence of two disulfide bonds. Disulfide bonds, however, are asignificant disadvantage to such molecules in that they can be brokenunder reducing conditions and must be properly formed in order to have auseful protein structure. Further, the size of the loops in Tendamistatare relatively small, thus limiting the size of the inserts that can beaccommodated in the scaffold. Moreover, it is well known that formingcorrect disulfide bonds in newly synthesized peptides is notstraightforward. When a protein is expressed in the cytoplasmic space ofE. coli, the most common host bacterium for protein overexpression,disulfide bonds are usually not formed, potentially making it difficultto prepare large quantities of engineered molecules.

Thus, there is an on-going need for small, single-chain artificialantibodies for a variety of therapeutic, diagnostic and catalyticapplications. In particular, there is an on-going need for artificialantibodies that are structurally stable at neutral pH.

SUMMARY OF THE INVENTION

The present invention provides a fibronectin type III (Fn3) molecule,wherein the Fn3 contains a stabilizing mutation. A stabilizing mutationis defined herein as a modification or change in the amino acid sequenceof the Fn3 molecule, such as a substitution of one amino acid foranother, that increases the melting point of the molecule by more than0.1° C. as compared to a molecule that is identical except for thechange. Alternatively, the change may increase the melting point by morethan 0.5° C. or even 1.0° C. or more. A method for determining themelting point of Fn3 molecules is given in Example 19 below.

The Fn3 may have at least one aspartic acid (Asp) residue and/or atleast one glutamic acid (Glu) residue that has been deleted orsubstituted with at least one other amino acid residue. For example, Asp7 and/or Asp 23 and/or Glu 9, may have been deleted or substituted withat least one other amino acid residue. Asp 7, Asp 23, or Glu 9, may havebeen substituted with an asparagine (Asn) or lysine (Lys) residue. Thepresent invention further provides an isolated nucleic acid molecule andan expression vector encoding an Fn3 molecule wherein the Fn3 contains astabilizing mutation.

The invention provides a fibronectin type III (Fn3) polypeptide monobodycontaining a plurality of Fn3 β-strand domain sequences that are linkedto a plurality of loop region sequences wherein the Fn3 contains astabilizing mutation. One or more of the monobody loop region sequencesof the Fn3 polypeptide vary by deletion, insertion or replacement of atleast two amino acids from the corresponding loop region sequences inwild-type Fn3. The β-strand domains of the monobody have at least about50% total amino acid sequence homology to the corresponding amino acidsequence of wild-type Fn3's β-strand domain sequences. Preferably, oneor more of the loop regions of the monobody contain amino acid residues:

i) from 15 to 16 inclusive in an AB loop;

ii) from 22 to 30 inclusive in a BC loop;

iii) from 39 to 45 inclusive in a CD loop;

iv) from 51 to 55 inclusive in a DE loop;

v) from 60 to 66 inclusive in an EF loop; and

vi) from 76 to 87 inclusive in an FG loop.

The invention also provides a nucleic acid molecule encoding a Fn3polypeptide monobody wherein the Fn3 contains a stabilizing mutation, aswell as an expression vector containing the nucleic acid molecule and ahost cell containing the vector.

The invention further provides a method of preparing a Fn3 polypeptidemonobody wherein the Fn3 contains a stabilizing mutation. The methodincludes providing a DNA sequence encoding a plurality of Fn3 β-stranddomain sequences that are linked to a plurality of loop regionsequences, wherein at least one loop region of the sequence contains aunique restriction enzyme site. The DNA sequence is cleaved at theunique restriction site. Then a preselected DNA segment is inserted intothe restriction site. The preselected DNA segment encodes a peptidecapable of binding to a specific binding partner (SBP) or a transitionstate analog compound (TSAC). The insertion of the preselected DNAsegment into the DNA sequence yields a DNA molecule which encodes apolypeptide monobody having an insertion. The DNA molecule is thenexpressed so as to yield the polypeptide monobody.

Also provided is a method of preparing a Fn3 polypeptide monobodywherein the Fn3 contains a stabilizing mutation, which method includesproviding a replicatable DNA sequence encoding a plurality of Fn3β-strand domain sequences that are linked to a plurality of loop regionsequences, wherein the nucleotide sequence of at least one loop regionis known. Polymerase chain reaction (PCR) primers are provided orprepared which are sufficiently complementary to the known loop sequenceso as to be hybridizable under PCR conditions, wherein at least one ofthe primers contains a modified nucleic acid sequence to be insertedinto the DNA sequence. PCR is performed using the replicatable DNAsequence and the primers. The reaction product of the PCR is thenexpressed so as to yield a polypeptide monobody.

The invention provides a further method of preparing a Fn3 polypeptidemonobody wherein the Fn3 contains a stabilizing mutation. The methodincludes providing a replicatable DNA sequence encoding a plurality ofFn3 β-strand domain sequences that are linked to a plurality of loopregion sequences, wherein the nucleotide sequence of at least one loopregion is known. Site-directed mutagenesis of at least one loop regionis performed so as to create an insertion mutation. The resultant DNAincluding the insertion mutation is then expressed.

Further provided is a variegated nucleic acid library encoding Fn3polypeptide monobodies including a plurality of nucleic acid speciesencoding a plurality of Fn3 β-strand domain sequences that are linked toa plurality of loop region sequences, wherein one or more of themonobody loop region sequences vary by deletion, insertion orreplacement of at least two amino acids from corresponding loop regionsequences in wild-type Fn3, and wherein the β-strand domains of themonobody have at least a 50% total amino acid sequence homology to thecorresponding amino acid sequence of β-strand domain sequences of thewild-type Fn3, and wherein the Fn3 contains a stabilizing mutation. Theinvention also provides a peptide display library derived from thevariegated nucleic acid library of the invention. Preferably, thepeptide of the peptide display library is displayed on the surface of abacteriophage, e.g., a M13 bacteriophage or a fd bacteriophage, orvirus.

The invention also provides a method of identifying the amino acidsequence of a polypeptide molecule capable of binding to a specificbinding partner (SBP) so as to form a polypeptide:SSP complex, whereinthe dissociation constant of the polypeptide:SBP complex is less than10⁻⁶ moles/liter. The method includes the steps of

-   -   a) providing a peptide display library of the invention;    -   b) contacting the peptide display library of (a) with an        immobilized or separable SBP;    -   c) separating the peptide:SBP complexes from the free peptides;    -   d) causing the replication of the separated peptides of (c) so        as to result in a new peptide display library distinguished from        that in (a) by having a lowered diversity and by being enriched        in displayed peptides capable of binding the SBP;    -   e) optionally repeating steps (b), (c), and (d) with the new        library of (d); and    -   f) determining the nucleic acid sequence of the region encoding        the displayed peptide of a species from (d) and hence deducing        the peptide sequence capable of binding to the SBP.

The present invention also provides a method of preparing a variegatednucleic acid library encoding Fn3 polypeptide monobodies having aplurality of nucleic acid species each including a plurality of loopregions, wherein the species encode a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein one or more of the loop region sequences vary by deletion,insertion or replacement of at least two amino acids from correspondingloop region sequences in wild-type Fn3, and wherein the β-strand domainsequences of the monobody have at least a 50% total amino acid sequencehomology to the corresponding amino acid sequences of β-strand domainsequences of the wild-type Fn3, and wherein the Fn3 contains astabilizing mutation, including the steps of

-   -   a) preparing an Fn3 polypeptide monobody having a predetermined        sequence;    -   b) contacting the polypeptide with a specific binding partner        (SBP) so as to form a polypeptide:SSP complex wherein the        dissociation constant of the polypeptide:SBP complex is less        than 10⁻⁶ moles/liter;    -   c) determining the binding structure of the polypeptide:SBP        complex by nuclear magnetic resonance spectroscopy or X-ray        crystallography; and    -   d) preparing the variegated nucleic acid library, wherein the        variegation is performed at positions in the nucleic acid        sequence which, from the information provided in (c), result in        one or more polypeptides with improved binding to the SBP.

Also provided is a method of identifying the amino acid sequence of apolypeptide molecule capable of catalyzing a chemical reaction with acatalyzed rate constant, k_(cat), and an uncatalyzed rate constant,k_(uncat), such that the ratio of k_(cat)/k_(uncat) greater than 10. Themethod includes the steps of:

-   -   a) providing a peptide display library of the invention;    -   b) contacting the peptide display library of (a) with an        immobilized or separable transition state analog compound (TSAC)        representing the approximate molecular transition state of the        chemical reaction;    -   c) separating the peptide:TSAC complexes from the free peptides;    -   d) causing the replication of the separated peptides of (c) so        as to result in a new peptide display library distinguished from        that in (a) by having a lowered diversity and by being enriched        in displayed peptides capable of binding the TSAC;    -   e) optionally repeating steps (b), (c), and (d) with the new        library of (d); and    -   f) determining the nucleic acid sequence of the region encoding        the displayed peptide of a species from (d) and hence deducing        the peptide sequence.

The invention also provides a method of preparing a variegated nucleicacid library encoding Fn3 polypeptide monobodies having a plurality ofnucleic acid species each including a plurality of loop regions, whereinthe species encode a plurality of Fn3 β-strand domain sequences that arelinked to a plurality of loop region sequences, wherein one or more ofthe loop region sequences vary by deletion, insertion or replacement ofat least two amino acids from corresponding loop region sequences inwild-type Fn3, and wherein the β-strand domain sequences of the monobodyhave at least a 50% total amino acid sequence homology to thecorresponding amino acid sequences of β-strand domain sequences of thewild-type Fn3, and wherein the Fn3 contains a stabilizing mutation,including the steps of

-   -   a) preparing an Fn3 polypeptide monobody having a predetermined        sequence, wherein the polypeptide is capable of catalyzing a        chemical reaction with a catalyzed rate constant, k_(cat), and        an uncatalyzed rate constant, k_(uncat), such that the ratio of        k_(cat)/k_(uncat) is greater than 10;    -   b) contacting the polypeptide with an immobilized or separable        transition state analog compound (TSAC) representing the        approximate molecular transition state of the chemical reaction;    -   c) determining the binding structure of the polypeptide:TSAC        complex by nuclear magnetic resonance spectroscopy or X-ray        crystallography; and    -   d) preparing the variegated nucleic acid library, wherein the        variegation is performed at positions in the nucleic acid        sequence which, from the information provided in (c), result in        one or more polypeptides with improved binding to or        stabilization of the TSAC.

The invention also provides a kit for the performance of any of themethods of the invention. The invention further provides a composition,e.g., a polypeptide, prepared by the use of the kit, or identified byany of the methods of the invention.

The following abbreviations have been used in describing amino acids,peptides, or proteins: Ala or A, Alanine; Arg or R, Arginine; Asn or Nasparagine; Asp or D, aspartic acid; Cys or C, cysteine; Gln or Q,glutamine; Glu or E, glutamic acid; Gly or G, glycine; His or H,histidine; Ile or I, isoleucine; Leu or L, leucine; Lys or K, lysine;Met or M, methionine; Phe or F, phenylalanine; Pro or P, proline; Ser orS, serine; Thr or T, threonine; Trp or W, tryptophan; Tyr or Y,tyrosine; Val or V, valine.

The following abbreviations have been used in describing nucleic acids,DNA, or RNA: A, adenosine; T, thymidine; G, guanosine; C, cytosine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. β-Strand and loop topology (A, B) and MOLSCRIPT representation(C, D; Kraulis, 1991) of the VH domain of anti-lysozyme immunoglobulinD1.3 (A, C; Bhat et al., 1994) and 10th type III domain of humanfibronectin (B, D; Main et al., 1992). The locations of complementaritydetermining regions (CDRs, hypervariable regions) and theintegrin-binding Arg-Gly-Asp (RGD) sequence are indicated.

FIG. 2. Amino acid sequence (SEQ ID NO:110) and restriction sites of thesynthetic Fn3 gene. The residue numbering is according to Main et al.(1992). Restriction enzyme sites designed are shown above the amino acidsequence. β-Strands are denoted by underlines. The N-terminal “mq”sequence has been added for a subsequent cloning into an expressionvector. The His.tag (Novagen) fusion protein has an additional sequence,MGSSHHHHHHSSGLVPRGSH (SEQ ID NO:114), preceding the Fn3 sequence shownabove.

FIG. 3. A, Far UV CD spectra of wild-type Fn3 at 25° C. and 90° C. Fn3(50 μM) was dissolved in sodium acetate (50 mM, pH 4.6). B, thermaldenaturation of Fn3 monitored at 215 nm. Temperature was increased at arate of 1° C./min.

FIG. 4. A, Cα trace of the crystal structure of the complex of lysozyme(HEL) and the Fv fragment of the anti-hen egg-white lysozyme (anti-HEL)antibody D1.3 (Bhat et al., 1994). Side chains of the residues 99-102 ofVH CDR3, which make contact with HEL, are also shown. B, Contact surfacearea for each residue of the D1.3 VH-HEL and VH-VL interactions plottedvs. residue number of D1.3 VH. Surface area and secondary structure weredetermined using the program DSSP (Kabsh and Sander, 1983). C and D,schematic drawings of the β-sheet structure of the F strand-loop-Gstrand moieties of D1.3 VH (C) and Fn3 (D). The boxes denote residues inβ-strands and ovals those not in strands. The shaded boxes indicateresidues of which side chains are significantly buried. The broken linesindicate hydrogen bonds.

FIG. 5. Designed Fn3 gene showing DNA (SEQ ID NO:111) and amino acid(SEQ ID NO:112) sequences. The amino acid numbering is according to Mainet al. (1992). The two loops that were randomized in combinatoriallibraries are enclosed in boxes.

FIG. 6. Map of plasmid pAS45. Plasmid pAS45 is the expression vector ofHis.tag-Fn3.

FIG. 7. Map of plasmid pAS25. Plasmid pAS25 is the expression vector ofFn3.

FIG. 8. Map of plasmid pAS38. pAS38 is a phagemid vector for the surfacedisplay of Fn3.

FIG. 9. (Ubiquitin-1) Characterization of ligand-specific binding ofenriched clones using phage enzyme-linked immunosolvent assay (ELISA).Microtiter plate wells were coated with ubiquitin (1 μg/well; “Ligand(+)) and then blocked with BSA. Phage solution in TBS containingapproximately 10¹⁰ colony forming units (cfu) was added to a well andwashed with TBS. Bound phages were detected with anti-phage antibody-PODconjugate (Pharmacia) with Turbo-TMB (Pierce) as a substrate. Absorbancewas measured using a Molecular Devices SPECTRAmax 250 microplatespectrophotometer. For a control, wells without the immobilized ligandwere used. 2-1 and 2-2 denote enriched clones from Library 2 eluted withfree ligand and acid, respectively. 4-1 and 4-2 denote enriched clonesfrom Library 4 eluted with free ligand and acid, respectively.

FIG. 10. (Ubiquitin-2) Competition phage ELISA of enriched clones.

Phage solutions containing approximately 10¹⁰ cfu were first incubatedwith free ubiquitin at 4° C. for 1 hour prior to the binding to aligand-coated well. The wells were washed and phages detected asdescribed above.

FIG. 11. Competition phage ELISA of ubiquitin-binding monobody 411.Experimental conditions are the same as described above for ubiquitin.The ELISA was performed in the presence of free ubiquitin in the bindingsolution. The experiments were performed with four differentpreparations of the same clone.

FIG. 12. (Fluorescein-1) Phage ELISA of four clones, Plb25.1 (containingSEQ ID NO:115), Plb25.4 (containing SEQ ID NO:116), pLB24.1 (containingSEQ ID NO:117) and pLB24.3 (containing SEQ ID NO:118). Experimentalconditions are the same as ubiquitin-1 above.

FIG. 13. (Fluorescein-2) Competition ELISA of the four clones.Experimental conditions are the same as ubiquitin-2 above.

FIG. 14. ¹H, ¹⁵N-HSQC spectrum of a fluorescence-binding monobodyLB25.5. Approximately 20 μM protein was dissolved in 10 mM sodiumacetate buffer (pH 5.0) containing 100 mM sodium chloride. The spectrumwas collected at 30° C. on a Varian Unity INOVA 600 NMR spectrometer.

FIG. 15. Characterization of the binding reaction of Ubi4-Fn3 to thetarget, ubiquitin. (a) Phage ELISA analysis of binding of Ubi4-Fn3 toubiquitin. The binding of Ubi4-phages to ubiquitin-coated wells wasmeasured. The control experiment was performed with wells containing noubiquitin.

(b) Competition phage ELISA of Ubi4-Fn3. Ubi4-Fn3-phages werepreincubated with soluble ubiquitin at an indicated concentration,followed by the phage ELISA detection in ubiquitin-coated wells.

(c) Competition phage ELISA testing the specificity of the Ubi4 clone.The Ubi4 phages were preincubated with 250 μg/ml of soluble proteins,followed by phage ELISA as in (b).

(d) ELISA using free proteins.

FIG. 16. Equilibrium unfolding curves for Ubi4-Fn3 (closed symbols) andwild-type Fn3 (open symbols). Squares indicate data measured in TBS(Tris HCl buffer (50 mM, pH 7.5) containing NaCl (150 mM)). Circlesindicate data measured in Gly HCl buffer (20 mM, pH 3.3) containing NaCl(300 mM). The curves show the best fit of the transition curve based onthe two-state model. Parameters characterizing the transitions arelisted in Table 8.

FIG. 17. (a) ¹H, ¹⁵N-HSQC spectrum of [¹⁵N]-Ubi4-K Fn3. (b). Difference(δ_(wild-type)-δ_(Ubi4)) of ¹H (b) and ¹⁵N (c) chemical shifts plottedversus residue number. Values for residues 82-84 (shown as filledcircles) where Ubi4-K deletions are set to zero. Open circles indicateresidues that are mutated in the Ubi4-K protein. The locations ofβ-strands are indicated with arrows.

FIG. 18. (A) Guanidine hydrochloride (GuHCl)-induced denaturation ofFNfn10 monitored by Trp fluorescence. The fluorescence emissionintensity at 355 nm is shown as a function of GuHCl concentration. Thelines show the best fits of the data to the two-state transition model.(B) Stability of FN3 at 4 M GuHCl plotted as a function of pH. (C) pHdependence of the m value.

FIG. 19. A two-dimensional H(C)CO spectrum of FNfn10 showing the ¹³Cchemical shift of the carboxyl carbon (vertical axis) and the ¹H shiftof ¹H^(β) of Asp or ¹H^(γ) of Glu, respectively (horizontal axis). Crosspeaks are labeled with their respective residue numbers.

FIG. 20. pH-Dependent shifts of the ¹³C chemical shifts of the carboxylcarbons of Asp and Glu residues in FNfn10. Panel A shows data for Asp 3,67 and 80, and Glu 38 and 47. The lines are the best fits of the data tothe Henderson-Hasselbalch equation with one ionizable group (McIntosh,L. P., Hand, G., Johnson, P. E., Joshi, M. D., Koerner, M., Plesniak, L.A., Ziser, L., Wakarchuk, W. W. & Withers, S. G. (1996) Biochemistry 35,9958-9966). Panel B shows data for Asp 7 and 23 and Glu 9. Thecontinuous lines show the best fits to the Henderson-Hasselbalchequation with two ionizable groups, while the dashed lines show the bestfits to the equation with a single ionizable group.

FIG. 21. (A) The amino acid sequence of FNfn10 (SEQ ID NO:121) shownaccording to its topology (Main, A. L., Harvey, T. S., Baron, M., Boyd,J., & Campbell, I. D. (1992) Cell 71, 671-678). Asp and Glu residues arehighlighted with gray circles. The thin lines and arrows connectingcircles indicate backbone hydrogen bonds. (B) A CPK model of FN3 showingthe locations of Asp 7 and 23 and Glu 9.

FIG. 22. Thermal denaturation of the wild-type and mutant FNfn10proteins at pH 7.0 and 2.4 in the presence of 6.3 M urea and 0.1 or 1.0M NaCl. Change in circular dichroism signal at 227 nm is plotted as afunction of temperature. The filled circles show the data in thepresence of 1 M NaCl and the open circles are data in the presence of0.1 M NaCl. The left column shows data taken at pH 2.4 and the rightcolumn at pH 7.0. The identity of proteins is indicated in the panels.

FIG. 23. GuHCl-induce denaturation of FNfn10 mutants monitored withfluorescence. Fluorescence data was converted to the fraction ofunfolded protein according to the two-state transition model (Loladze,V. V., Ibarra-Molero, B., Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999)Biochemistry 38, 16419-16423), and plotted as a function of GuHCl.

FIG. 24. pH Titration of the carboxyl ¹³C resonance of Asp and Gluresidues in D7N (open circles) and D7K (closed circles) FNfn10. Data forthe wild-type (crosses) are also shown for comparison. Residue names aredenoted in the individual panels.

DETAILED DESCRIPTION OF THE INVENTION

For the past decade the immune system has been exploited as a richsource of de novo catalysts. Catalytic antibodies have been shown tohave chemoselectivity, enantioselectivity, large rate accelerations, andeven an ability to reroute chemical reactions. In most cases theantibodies have been elicited to transition state analog (TSA) haptens.These TSA haptens are stable, low-molecular weight compounds designed tomimic the structures of the energetically unstable transition statespecies that briefly (approximate half-life 10⁻¹³ s) appear alongreaction pathways between reactants and products. Anti-TSA antibodies,like natural enzymes, are thought to selectively bind and stabilizetransition state, thereby easing the passage of reactants to products.Thus, upon binding, the antibody lowers the energy of the actualtransition state and increases the rate of the reaction. These catalystscan be programmed to bind to geometrical and electrostatic features ofthe transition state so that the reaction route can be controlled byneutralizing unfavorable charges, overcoming entropic barriers, anddictating stereoelectronic features of the reaction. By this means evenreactions that are otherwise highly disfavored have been catalyzed(Janda et al. 1997). Further, in many instances catalysts have been madefor reactions for which there are no known natural or man-made enzymes.

The success of any combinatorial chemical system in obtaining aparticular function depends on the size of the library and the abilityto access its members. Most often the antibodies that are made in ananimal against a hapten that mimics the transition state of a reactionare first screened for binding to the hapten and then screened again forcatalytic activity. An improved method allows for the direct selectionfor catalysis from antibody libraries in phage, thereby linkingchemistry and replication.

A library of antibody fragments can be created on the surface offilamentous phage viruses by adding randomized antibody genes to thegene that encodes the phage's coat protein. Each phage then expressesand displays multiple copies of a single antibody fragment on itssurface. Because each phage possesses both the surface-displayedantibody fragment and the DNA that encodes that fragment, and antibodyfragment that binds to a target can be identified by amplifying theassociated DNA.

Immunochemists use as antigens materials that have as little chemicalreactivity as possible. It is almost always the case that one wishes theultimate antibody to interact with native structures. In reactiveimmunization the concept is just the opposite. One immunizes withcompounds that are highly reactive so that upon binding to the antibodymolecule during the induction process, a chemical reaction ensues. Laterthis same chemical reaction becomes part of the mechanism of thecatalytic event. In a certain sense one is immunizing with a chemicalreaction rather than a substance per se. Reactive immunogens can beconsidered as analogous to the mechanism-based inhibitors thatenzymologists use except that they are used in the inverse way in that,instead of inhibiting a mechanism, they induce a mechanism.

Man-made catalytic antibodies have considerable commercial potential inmany different applications. Catalytic antibody-based products have beenused successfully in prototype experiments in therapeutic applications,such as prodrug activation and cocaine inactivation, and innontherapeutic applications, such as biosensors and organic synthesis.

Catalytic antibodies are theoretically more attractive than noncatalyticantibodies as therapeutic agents because, being catalytic, they may beused in lower doses, and also because their effects are unusuallyirreversible (for example, peptide bond cleavage rather than binding).In therapy, purified catalytic antibodies could be directly administeredto a patient, or alternatively the patient's own catalytic antibodyresponse could be elicited by immunization with an appropriate hapten.Catalytic antibodies also could be used as clinical diagnostic tools oras regioselective or stereoselective catalysts in the synthesis of finechemicals.

I. Mutation of Fn3 Loops and Grafting of Ab Loops onto Fn3

An ideal scaffold for CDR grafting is highly soluble and stable. It issmall enough for structural analysis, yet large enough to accommodatemultiple CDRs so as to achieve tight binding and/or high specificity.

A novel strategy to generate an artificial Ab system on the framework ofan existing non-Ab protein was developed. An advantage of this approachover the minimization of an Ab scaffold is that one can avoid inheritingthe undesired properties of Abs. Fibronectin type III domain (Fn3) wasused as the scaffold. Fibronectin is a large protein which playsessential roles in the formation of extracellular matrix and cell-cellinteractions; it consists of many repeats of three types (I, II and III)of small domains (Baron et al., 1991). Fn3 itself is the paradigm of alarge subfamily (Fn3 family or s-type Ig family) of the immunoglobulinsuperfamily (IgSF). The Fn3 family includes cell adhesion molecules,cell surface hormone and cytokine receptors, chaperonins, andcarbohydrate-binding domains (for reviews, see Bork & Doolittle, 1992;Jones, 1993; Bork et al., 1994; Campbell & Spitzfaden, 1994; Harpez &Chothia, 1994).

Recently, crystallographic studies revealed that the structure of theDNA binding domains of the transcription factor NF-kB is also closelyrelated to the Fn3 fold (Ghosh et al., 1995; Müller et al., 1995). Theseproteins are all involved in specific molecular recognition, and in mostcases ligand-binding sites are formed by surface loops, suggesting thatthe Fn3 scaffold is an excellent framework for building specific bindingproteins. The 3D structure of Fn3 has been determined by NMR (Main etal., 1992) and by X-ray crystallography (Leahy et al., 1992; Dickinsonet al., 1994). The structure is best described as a β-sandwich similarto that of Ab VH domain except that Fn3 has seven β-strands instead ofnine (FIG. 1). There are three loops on each end of Fn3; the positionsof the BC, DE and FG loops approximately correspond to those of CDR1, 2and 3 of the VH domain, respectively (FIG. 1 C, D).

Fn3 is small (˜95 residues), monomeric, soluble and stable. It is one offew members of IgSF that do not have disulfide bonds; VH has aninterstrand disulfide bond (FIG. 1 A) and has marginal stability underreducing conditions. Fn3 has been expressed in E. coli (Aukhil et al.,1993). In addition, 17 Fn3 domains are present just in humanfibronectin, providing important information on conserved residues whichare often important for the stability and folding (for sequencealignment, see Main et al., 1992 and Dickinson et al., 1994). Fromsequence analysis, large variations are seen in the BC and FG loops,suggesting that the loops are not crucial to stability. NMR studies haverevealed that the FG loop is highly flexible; the flexibility has beenimplicated for the specific binding of the 10th Fn3 to α₅β₁ integrinthrough the Arg-Gly-Asp (RGD) motif. In the crystal structure of humangrowth hormone-receptor complex (de Vos et al., 1992), the second Fn3domain of the receptor interacts with hormone via the FG and BC loops,suggesting it is feasible to build a binding site using the two loops.

The tenth type III module of fibronectin has a fold similar to that ofimmunoglobulin domains, with seven β strands forming two antiparallel βsheets, which pack against each other (Main et al., 1992). The structureof the type II module consists of seven β strands, which form a sandwichof two antiparallel β sheets, one containing three strands (ABE) and theother four strands (C′CFG) (Williams et al., 1988). The triple-strandedβ sheet consists of residues Glu-9-Thr-14 (A), Ser-17-Asp-23 (B), andThr-56-Ser-60 (E). The majority of the conserved residues contribute tothe hydrophobic core, with the invariant hydrophobic residues Trp-22 andTry-68 lying toward the N-terminal and C-terminal ends of the core,respectively. The β strands are much less flexible and appear to providea rigid framework upon which functional, flexible loops are built. Thetopology is similar to that of immunoglobulin C domains.

Gene Construction and Mutagenesis

A synthetic gene for tenth Fn3 of human fibronectin (FIG. 2) wasdesigned which includes convenient restriction sites for ease ofmutagenesis and uses specific codons for high-level protein expression(Gribskov et al., 1984).

The gene was assembled as follows: (1) the gene sequence was dividedinto five parts with boundaries at designed restriction sites (FIG. 2);(2) for each part, a pair of oligonucleotides that code opposite strandsand have complementary overlaps of ˜15 bases was synthesized; (3) thetwo oligonucleotides were annealed and single strand regions were filledin using the Klenow fragment of DNA polymerase; (4) the double-strandedoligonucleotide was cloned into the pET3a vector (Novagen) usingrestriction enzyme sites at the termini of the fragment and its sequencewas confirmed by an Applied Biosystems DNA sequencer using the dideoxytermination protocol provided by the manufacturer; (5) steps 2-4 wererepeated to obtain the whole gene (plasmid pAS25) (FIG. 7).

Although the present method takes more time to assemble a gene than theone-step polymerase chain reaction (PCR) method (Sandhu et al., 1992),no mutations occurred in the gene. Mutations would likely have beenintroduced by the low fidelity replication by Taq polymerase and wouldhave required time-consuming gene editing. The gene was also cloned intothe pET15b (Novagen) vector (pEW1). Both vectors expressed the Fn3 geneunder the control of bacteriophage T7 promoter (Studler et al. 1990);pAS25 expressed the 96-residue Fn3 protein only, while pEW1 expressedFn3 as a fusion protein with poly-histidine peptide (His.tag).Recombinant DNA manipulations were performed according to MolecularCloning (Sambrook et al., 1989), unless otherwise stated.

Mutations were introduced to the Fn3 gene using either cassettemutagenesis or oligonucleotide site-directed mutagenesis techniques(Deng & Nickoloff, 1992). Cassette mutagenesis was performed using thesame protocol for gene construction described above; double-stranded DNAfragment coding a new sequence was cloned into an expression vector(pAS25 and/or pEW1). Many mutations can be made by combining a newlysynthesized strand (coding mutations) and an oligonucleotide used forthe gene synthesis. The resulting genes were sequenced to confirm thatthe designed mutations and no other mutations were introduced bymutagenesis reactions.

Design and Synthesis of Fn3 Mutants with Antibody CDRs

Two candidate loops (FG and BC) were identified for grafting. Antibodieswith known crystal structures were examined in order to identifycandidates for the sources of loops to be grafted onto Fn3. Anti-hen egglysozyme (HEL) antibody D1.3 (Bhat et al., 1994) was chosen as thesource of a CDR loop. The reasons for this choice were: (1) highresolution crystal structures of the free and complexed states areavailable (FIG. 4 A; Bhat et al., 1994), (2) thermodynamics data for thebinding reaction are available (Tello et al., 1993), (3) D1.3 has beenused as a paradigm for Ab structural analysis and Ab engineering(Verhoeyen et al., 1988; McCafferty et al., 1990) (4) site-directedmutagenesis experiments have shown that CDR3 of the heavy chain(VH-CDR3) makes a larger contribution to the affinity than the otherCDRs (Hawkins et al., 1993), and (5) a binding assay can be easilyperformed. The objective for this trial was to graft VH-CDR3 of D1.3onto the Fn3 scaffold without significant loss of stability.

An analysis of the D1.3 structure (FIG. 4) revealed that only residues99-102 (“RDYR”) (SEQ ID NO:120) make direct contact with hen egg-whitelysozyme (HEL) (FIG. 4 B), although VH-CDR3 is defined as longer (Bhatet al., 1994). It should be noted that the C-terminal half of VH-CDR3(residues 101-104) made significant contact with the VL domain (FIG. 4B). It has also become clear that D1.3 VH-CDR3 (FIG. 4 C) has a shorterturn between the strands F and G than the FG loop of Fn3 (FIG. 4 D).Therefore, mutant sequences were designed by using the RDYR (99-102)(SEQ ID NO:120) of D1.3 as the core and made different boundaries andloop lengths (Table 1). Shorter loops may mimic the D1.3 CDR3conformation better, thereby yielding higher affinity, but they may alsosignificantly reduce stability by removing wild-type interactions ofFn3.

TABLE 1 Amino acid sequences of D1.3 VH CDR3, VH8 CDR3 andFn3 FG loop and list of planned mutants. D1.3 96 100   105(SEQ ID NO: 1) •   •     • ARERDYRLDYWGQG VH8 ARGAVVSYYAMDYWGQG(SEQ ID NO: 2) Fn3   75   80   85 (SEQ ID NO: 3)    •    •    •YAVTGRGDSPASSKPI Mutant Sequence D1.3-1 YAERDYRLDY----PI (SEQ ID NO: 4)D1.3-2 YAVRDYRLDY----PI (SEQ ID NO: 5) D1.3-3 YAVRDYRLDYASSKPI(SEQ ID NO: 6) D1.3-4 YAVRDYRLDY---KPI (SEQ ID NO: 7) D1.3-5YAVRDYR-----SKPI (SEQ ID NO: 8) D1.3-6 YAVTRDYRL--SSKPI (SEQ ID NO: 9)D1.3-7 YAVTERDYRL-SSKPI (SEQ ID NO: 10) VH8-1 YAVAVVSYYAMDY-PI(SEQ ID NO: 11) VH8-2 YAVTAVVSYYASSKPI (SEQ ID NO: 12) Underlinesindicate residues in β-strands. Bold characters indicate replacedresidues.

In addition, an anti-HEL single VH domain termed VH8 (Ward et al., 1989)was chosen as a template. VH8 was selected by library screening and, inspite of the lack of the VL domain, VH8 has an affinity for HEL of 27nM, probably due to its longer VH-CDR3 (Table 1). Therefore, its VH-CDR3was grafted onto Fn3. Longer loops may be advantageous on the Fn3framework because they may provide higher affinity and also are close tothe loop length of wild-type Fn3. The 3D structure of VH8 was not knownand thus the VH8 CDR3 sequence was aligned with that of D1.3 VH-CDR3;two loops were designed (Table 1).

Mutant Construction and Production

Site-directed mutagenesis experiments were performed to obtain designedsequences. Two mutant Fn3s, D1.3-1 and D1.3-4 (Table 1) were obtainedand both were expressed as soluble His.tag fusion proteins. D1.3-4 waspurified and the His.tag portion was removed by thrombin cleavage.D1.3-4 is soluble up to at least 1 mM at pH 7.2. No aggregation of theprotein has been observed during sample preparation and NMR dataacquisition.

Protein Expression and Purification

E. coli BL21 (DE3) (Novagen) were transformed with an expression vector(pAS25, pEW1 and their derivatives) containing a gene for the wild-typeor a mutant. Cells were grown in M9 minimal medium and M9 mediumsupplemented with Bactotrypton (Difco) containing ampicillin (200μg/ml). For isotopic labeling, ¹⁵N NH₄Cl and/or ¹³C glucose replacedunlabeled components. 500 ml medium in a 2 liter baffle flask wereinoculated with 10 ml of overnight culture and agitated at 37° C.Isopropylthio-β-galactoside (IPTG) was added at a final concentration of1 mM to initiate protein expression when OD (600 nm) reaches one. Thecells were harvested by centrifugation 3 hours after the addition ofIPTG and kept frozen at −70° C. until used.

Fn3 without His.tag was purified as follows. Cells were suspended in 5ml/(g cell) of Tris (50 mM, pH 7.6) containingethylenediaminetetraacetic acid (EDTA; 1 mM) and phenylmethylsulfonylfluoride (1 mM). HEL was added to a final concentration of 0.5 mg/ml.After incubating the solution for 30 minutes at 37° C., it was sonicatedthree times for 30 seconds on ice. Cell debris was removed bycentrifugation. Ammonium sulfate was added to the solution andprecipitate recovered by centrifugation. The pellet was dissolved in5-10 ml sodium acetate (50 mM, pH 4.6) and insoluble material wasremoved by centrifugation. The solution was applied to a SephacrylS100HR column (Pharmacia) equilibrated in the sodium acetate buffer.Fractions containing Fn3 then was applied to a ResourceS column(Pharmacia) equilibrated in sodium acetate (50 mM, pH 4.6) and elutedwith a linear gradient of sodium chloride (0-0.5 M). The protocol can beadjusted to purify mutant proteins with different surface chargeproperties.

Fn3 with His.tag was purified as follows. The soluble fraction wasprepared as described above, except that sodium phosphate buffer (50 mM,pH 7.6) containing sodium chloride (100 mM) replaced the Tris buffer.The solution was applied to a Hi-Trap chelating column (Pharmacia)preloaded with nickel and equilibrated in the phosphate buffer. Afterwashing the column with the buffer, His.tag-Fn3 was eluted in thephosphate buffer containing 50 mM EDTA. Fractions containing His.tag-Fn3were pooled and applied to a Sephacryl S100-HR column, yielding highlypure protein. The His.tag portion was cleaved off by treating the fusionprotein with thrombin using the protocol supplied by Novagen. Fn3 wasseparated from the His.tag peptide and thrombin by a ResourceS columnusing the protocol above.

The wild-type and two mutant proteins so far examined are expressed assoluble proteins. In the case that a mutant is expressed as inclusionbodies (insoluble aggregate), it is first examined if it can beexpressed as a soluble protein at lower temperature (e.g., 25-30° C.).If this is not possible, the inclusion bodies are collected by low-speedcentrifugation following cell lysis as described above. The pellet iswashed with buffer, sonicated and centrifuged. The inclusion bodies aresolubilized in phosphate buffer (50 mM, pH 7.6) containing guanidiniumchloride (GdnCl, 6 M) and will be loaded on a Hi-Trap chelating column.The protein is eluted with the buffer containing GdnCl and 50 mM EDTA.

Conformation of Mutant Fn3, D1.3-4

The ¹H NMR spectra of His.tag D1.3-4 fusion protein closely resembledthat of the wild-type, suggesting the mutant is folded in a similarconformation to that of the wild-type. The spectrum of D1.3-4 after theremoval of the His.tag peptide showed a large spectral dispersion. Alarge dispersion of amide protons (7-9.5 ppm) and a large number ofdownfield (5.0-6.5 ppm) C^(α) protons are characteristic of a β-sheetprotein (Wüthrich, 1986).

The 2D NOESY spectrum of D1.3-4 provided further evidence for apreserved conformation. The region in the spectrum showed interactionsbetween upfield methyl protons (<0.5 ppm) and methyl-methylene protons.The Val72 γ methyl resonances were well separated in the wild-typespectrum (−0.07 and 0.37 ppm; (Baron et al., 1992)). Resonancescorresponding to the two methyl protons are present in the D1.3-4spectrum (−0.07 and 0.44 ppm). The cross peak between these tworesonances and other conserved cross peaks indicate that the tworesonances in the D1.3-4 spectrum are highly likely those of Val72 andthat other methyl protons are in nearly identical environment to that ofwild-type Fn3. Minor differences between the two spectra are presumablydue to small structural perturbation due to the mutations. Val72 is onthe F strand, where it forms a part of the central hydrophobic core ofFn3 (Main et al., 1992). It is only four residues away from the mutatedresidues of the FG loop (Table 1). The results are remarkable because,despite there being 7 mutations and 3 deletions in the loop (more than10% of total residues; FIG. 12, Table 2), D1.3-4 retains a 3D structurevirtually identical to that of the wild-type (except for the mutatedloop). Therefore, the results provide strong support that the FG loop isnot significantly contributing to the folding and stability of the Fn3molecule and thus that the FG loop can be mutated extensively.

TABLE 2 Sequences of oligonucleotides Name Sequence FN1FCGGGATCCCATATGCAGGTTTCTGATGTTCCGCGTGACCTGGAAGTTGTTGCTGCGACC (SEQ ID NO: 13) FN1RTAACTGCAGGAGCATCCCAGCTGATCAGCAGGCTAGTC GGGGTCGCAGCAACAAC (SEQ ID NO: 14)FN2F CTCCTGCAGTTACCGTGCGTTATTACCGTATCACGTACGGTGAAACCGGTG (SEQ ID NO: 15) FN2R GTGAATTCCTGAACCGGGGAGTTACCACCGGTTTCACCG (SEQ ID NO: 16) FN3F AGGAATTCACTGTACCTGGTTCCAAGTCTACTGCTACCATCAGCGG (SEQ ID NO: 17) FN3R GTATAGTCGACACCCGGTTTCAGGCCGCTGATGGTAGC(SEQ ID NO: 18) FN4F CGGGTGTCGACTATACCATCACTGTATACGCT (SEQ ID NO: 19)FN4R CGGGATCCGAGCTCGCTGGGCTGTCACCACGGCCAGTAACAGCGTATACAGTGAT (SEQ ID NO: 20) FN5FCAGCGAGCTCCAAGCCAATCTCGATTAACTACCGT (SEQ ID NO: 21) FN5RCGGGATCCTCGAGTTACTAGGTACGGTAGTTAATCGA (SEQ ID NO: 22) FN5R′CGGGATCCACGCGTGCCACCGGTACGGTAGTTAATCGA (SEQ ID NO: 23) gene3FCGGGATCCACGCGTCCATTCGTTTGTGAATATCAAGGCC AATCG (SEQ ID NO: 24) gene3RCCGGAAGCTTTAAGACTCCTTATTACGCAGTATGTTAGC (SEQ ID NO: 25) 38TAABglIICTGTTACTGGCCGTGAGATCTAACCAGCGAGCTCCA (SEQ ID NO: 26) BC3GATCAGCTGGGATGCTCCTNNKNNKNNKNNKNNKTATT ACCGTATCACGTA (SEQ ID NO: 27) FG2TGTATACGCTGTTACTGGCNNKNNKNNKNNKNNKNNKNNKTCCAAGCCAATCTCGAT (SEQ ID NO: 28) FG3CTGTATACGCTGTTACTGGCNNKNNKNNKNNKCCAGCG AGCTCCAAG (SEQ ID NO: 29) FG4CATCACTGTATACGCTGTTACTNNKNNKNNKNNKNNKT CCAAGCCAATCTC (SEQ ID NO: 30)Restriction enzyme sites are underlined. N and K denote an equimolarmixture of A, T. G and C and that of G and T, respectively.

Structure and Stability Measurements

Structures of Abs were analyzed using quantitative methods (e.g., DSSP(Kabsch & Sander, 1983) and PDBfit (D. McRee, The Scripps ResearchInstitute)) as well as computer graphics (e.g., Quanta (MolecularSimulations) and What if (G. Vriend, European Molecular BiologyLaboratory)) to superimpose the strand-loop-strand structures of Abs andFn3.

The stability of monobodies was determined by measuring temperature- andchemical denaturant-induced unfolding reactions (Pace et al., 1989). Thetemperature-induced unfolding reaction was measured using a circulardichroism (CD) polarimeter. Ellipticity at 222 and 215 nm was recordedas the sample temperature was slowly raised. Sample concentrationsbetween 10 and 50 μM were used. After the unfolding baseline wasestablished, the temperature was lowered to examine the reversibility ofthe unfolding reaction. Free energy of unfolding was determined byfitting data to the equation for the two-state transition (Becktel &Schellman, 1987; Pace et al., 1989). Nonlinear least-squares fitting wasperformed using the program Igor (WaveMetrics) on a Macintosh computer.

The structure and stability of two selected mutant Fn3s were studied;the first mutant was D1.3-4 (Table 2) and the second was a mutant calledAS40 which contains four mutations in the BC loop (A²⁶V²⁷T²⁸V²⁹)→TQRQ).AS40 was randomly chosen from the BC loop library described above. Bothmutants were expressed as soluble proteins in E. coli and wereconcentrated at least to 1 mM, permitting NMR studies.

The mid-point of the thermal denaturation for both mutants wasapproximately 69° C., as compared to approximately 79° C. for thewild-type protein. The results indicated that the extensive mutations atthe two surface loops did not drastically decrease the stability of Fn3,and thus demonstrated the feasibility of introducing a large number ofmutations in both loops.

Stability was also determined by guanidinium chloride (GdnCl)- andurea-induced unfolding reactions. Preliminary unfolding curves wererecorded using a fluorometer equipped with a motor-driven syringe; GdnClor urea were added continuously to the protein solution in the cuvette.Based on the preliminary unfolding curves, separate samples containingvarying concentration of a denaturant were prepared and fluorescence(excitation at 290 nm, emission at 300-400 nm) or CD (ellipticity at 222and 215 nm) were measured after the samples were equilibrated at themeasurement temperature for at least one hour. The curve was fitted bythe least-squares method to the equation for the two-state model(Santoro & Bolen, 1988; Koide et al., 1993). The change in proteinconcentration was compensated if required.

Once the reversibility of the thermal unfolding reaction is established,the unfolding reaction is measured by a Microcal MC-2 differentialscanning calorimeter (DSC). The cell (˜1.3 ml) will be filled with FnAbsolution (0.1-1 mM) and ΔCp (=ΔH/ΔT) will be recorded as the temperatureis slowly raised. T_(m) (the midpoint of unfolding), ΔH of unfolding andΔG of unfolding is determined by fitting the transition curve (Privalov& Potekhin, 1986) with the Origin software provided by Microcal.

Thermal Unfolding

A temperature-induced unfolding experiment on Fn3 was performed usingcircular dichroism (CD) spectroscopy to monitor changes in secondarystructure. The CD spectrum of the native Fn3 shows a weak signal near222 nm (FIG. 3A), consistent with the predominantly β-structure of Fn3(Perczel et al., 1992). A cooperative unfolding transition is observedat 80-90° C., clearly indicating high stability of Fn3 (FIG. 3B). Thefree energy of unfolding could not be determined due to the lack of apost-transition baseline. The result is consistent with the highstability of the first Fn3 domain of human fibronectin (Litvinovich etal., 1992), thus indicating that Fn3 domains are in general highlystable.

Binding Assays

The binding reactions of monobodies were characterized quantitativelyusing an isothermal titration calorimeter (ITC) and fluorescencespectroscopy.

The enthalpy change (ΔH) of binding were measured using a Microcal OmegaITC (Wiseman et al., 1989). The sample cell (˜1.3 ml) was filled withMonobody solution (≦100 changed according to K_(d)), and the referencecell filled with distilled water; the system was equilibrated at a giventemperature until a stable baseline is obtained; 5-20 μl of ligandsolution (≦2 mM) was injected by a motor-driven syringe within a shortduration (20 sec) followed by an equilibration delay (4 minutes); theinjection was repeated and heat generation/absorption for each injectionwas measured. From the change in the observed heat change as a functionof ligand concentration, ΔH and K_(d) was determined (Wiseman et al.,1989). ΔG and ΔS of the binding reaction was deduced from the twodirectly measured parameters. Deviation from the theoretical curve wasexamined to assess nonspecific (multiple-site) binding. Experiments werealso be performed by placing a ligand in the cell and titrating with anFnAb. It should be emphasized that only ITC gives direct measurement ofΔH, thereby making it possible to evaluate enthalpic and entropiccontributions to the binding energy. ITC was successfully used tomonitor the binding reaction of the D1.3 Ab (Tello et al., 1993; Bhat etal., 1994).

Intrinsic fluorescence is monitored to measure binding reactions withK_(d) in the sub-μM range where the determination of K_(d) by ITC isdifficult. Trp fluorescence (excitation at ˜290 nm, emission at 300-350nm) and Tyr fluorescence (excitation at ˜260 nm, emission at ˜303 nm) ismonitored as the Fn3-mutant solution (≦10 μM) is titrated with ligandsolution (≦100 μM). K_(d) of the reaction is determined by the nonlinearleast-squares fitting of the bimolecular binding equation. Presence ofsecondary binding sites is examined using Scatchard analysis. In allbinding assays, control experiments are performed busing wild-type Fn3(or unrelated monobodies) in place of monobodies of interest.

II. Production of Fn3 Mutants with High Affinity and SpecificityMonobodies

Library screening was carried out in order to select monobodies thatbind to specific ligands. This is complementary to the modeling approachdescribed above. The advantage of combinatorial screening is that onecan easily produce and screen a large number of variants (≧10⁸), whichis not feasible with specific mutagenesis (“rational design”)approaches. The phage display technique (Smith, 1985; O'Neil & Hoess,1995) was used to effect the screening processes. Fn3 was fused to aphage coat protein (pIII) and displayed on the surface of filamentousphages. These phages harbor a single-stranded DNA genome that containsthe gene coding the Fn3 fusion protein. The amino acid sequence ofdefined regions of Fn3 were randomized using a degenerate nucleotidesequence, thereby constructing a library. Phages displaying Fn3 mutantswith desired binding capabilities were selected in vitro, recovered andamplified. The amino acid sequence of a selected clone can be identifiedreadily by sequencing the Fn3 gene of the selected phage. The protocolsof Smith (Smith & Scott, 1993) were followed with minor modifications.

The objective was to produce Monobodies which have high affinity tosmall protein ligands. HEL and the B1 domain of staphylococcal protein G(hereafter referred to as protein G) were used as ligands. Protein G issmall (56 amino acids) and highly stable (Minor & Kim, 1994; Smith etal., 1994). Its structure was determined by NMR spectroscopy (Gronenbornet al., 1991) to be a helix packed against a four-strand β-sheet. Theresulting FnAb-protein G complexes (˜150 residues) is one of thesmallest protein-protein complexes produced to date, well within therange of direct NMR methods. The small size, the high stability andsolubility of both components and the ability to label each with stableisotopes (¹³C and ¹⁵N; see below for protein G) make the complexes anideal model system for NMR studies on protein-protein interactions.

The successful loop replacement of Fn3 (the mutant D1.3-4) demonstratethat at least ten residues can be mutated without the loss of the globalfold. Based on this, a library was first constructed in which onlyresidues in the FG loop are randomized. After results of loopreplacement experiments on the BC loop were obtained, mutation siteswere extended that include the BC loop and other sites.

Construction of Fn3 Phage Display System

An M13 phage-based expression vector pASM1 has been constructed asfollows: an oligonucleotide coding the signal peptide of OmpT was clonedat the 5′ end of the Fn3 gene; a gene fragment coding the C-terminaldomain of M13 pIII was prepared from the wild-type gene III gene of M13mp18 using PCR (Corey et al., 1993) and the fragment was inserted at the3′ end of the OmpT-Fn3 gene; a spacer sequence has been inserted betweenFn3 and pIII. The resultant fragment (OmpT-Fn3-pIII) was cloned in themultiple cloning site of M13 mp18, where the fusion gene is under thecontrol of the lac promoter. This system will produce the Fn3-pIIIfusion protein as well as the wild-type pIII protein. The co-expressionof wild-type pIII is expected to reduce the number of fusion pIIIprotein, thereby increasing the phage infectivity (Corey et al., 1993)(five copies of pill are present on a phage particle). In addition, asmaller number of fusion pIII protein may be advantageous in selectingtight binding proteins, because the chelating effect due to multiplebinding sites should be smaller than that with all five copies of fusionpIII (Bass et al., 1990). This system has successfully displayed theserine protease trypsin (Corey et al., 1993). Phages were produced andpurified using E. coli K91kan (Smith & Scott, 1993) according to astandard method (Sambrook et al., 1989) except that phage particles werepurified by a second polyethylene glycol precipitation and acidprecipitation.

Successful display of Fn3 on fusion phages has been confirmed by ELISAusing an Ab against fibronectin (Sigma), clearly indicating that it isfeasible to construct libraries using this system.

An alternative system using the fUSE5 (Parmley & Smith, 1988) may alsobe used. The Fn3 gene is inserted to fUSE5 using the SfiI restrictionsites introduced at the 5′- and 3′-ends of the Fn3 gene PCR. This systemdisplays only the fusion pIII protein (up to five copies) on the surfaceof a phage. Phages are produced and purified as described (Smith &Scott, 1993). This system has been used to display many proteins and isrobust. The advantage of fUSE5 is its low toxicity. This is due to thelow copy number of the replication form (RF) in the host, which in turnmakes it difficult to prepare a sufficient amount of RF for libraryconstruction (Smith & Scott, 1993).

Construction of Libraries

The first library was constructed of the Fn3 domain displayed on thesurface of M13 phage in which seven residues (77-83) in the FG loop(FIG. 4D) were randomized. Randomization will be achieved by the use ofan oligonucleotide containing degenerated nucleotide sequence. Adouble-stranded nucleotide was prepared by the same protocol as for genesynthesis (see above) except that one strand had an (NNK)₆(NNG) sequenceat the mutation sites, where N corresponds to an equimolar mixture of A,T, G and C and K corresponds to an equimolar mixture of G and T. The(NNG) codon at residue 83 was required to conserve the Sad restrictionsite (FIG. 2). The (NNK) codon codes all of the 20 amino acids, whilethe NNG codon codes 14. Therefore, this library contained ˜10⁹independent sequences. The library was constructed by ligating thedouble-stranded nucleotide into the wild-type phage vector, pASM1, andthe transfecting E. coli XL1 blue (Stratagene) using electroporation.XL1 blue has the lacI^(q) phenotype and thus suppresses the expressionof the Fn3-pIII fusion protein in the absence of lac inducers. Theinitial library was propagated in this way, to avoid selection againsttoxic Fn3-pIII clones. Phages displaying the randomized Fn3-pIII fusionprotein were prepared by propagating phages with K91kan as the host.K91kan does not suppress the production of the fusion protein, becauseit does not have lacIq. Another library was also generated in which theBC loop (residues 26-20) was randomized.

Selection of Displayed Monobodies

Screening of Fn3 phage libraries was performed using the biopanningprotocol (Smith & Scott, 1993); a ligand is biotinylated and the strongbiotin-streptavidin interaction was used to immobilize the ligand on astreptavidin-coated dish. Experiments were performed at room temperature(˜22° C.). For the initial recovery of phages from a library, 10 μg of abiotinylated ligand were immobilized on a streptavidin-coatedpolystyrene dish (35 mm, Falcon 1008) and then a phage solution(containing ˜10″ pfu (plaque-forming unit)) was added. After washing thedish with an appropriate buffer (typically TBST, Tris-HCl (50 mM, pH7.5), NaCl (150 mM) and Tween 20 (0.5%)), bound phages were eluted byone or combinations of the following conditions: low pH, an addition ofa free ligand, urea (up to 6 M) and, in the case of anti-protein GMonobodies, cleaving the protein G-biotin linker by thrombin. Recoveredphages were amplified using the standard protocol using K91kan as thehost (Sambrook et al., 1989). The selection process were repeated 3-5times to concentrate positive clones. From the second round on, theamount of the ligand were gradually decreased (to ˜1 μg) and thebiotinylated ligand were mixed with a phage solution before transferringa dish (G. P. Smith, personal communication). After the final round,10-20 clones were picked, and their DNA sequence will be determined. Theligand affinity of the clones were measured first by the phage-ELISAmethod (see below).

To suppress potential binding of the Fn3 framework (background binding)to a ligand, wild-type Fn3 may be added as a competitor in the buffers.In addition, unrelated proteins (e.g., bovine serum albumin, cytochromec and RNase A) may be used as competitors to select highly specificMonobodies.

Binding Assay

The binding affinity of Monobodies on phage surface is characterizedsemi-quantitatively using the phage ELISA technique (Li et al., 1995).Wells of microtiter plates (Nunc) are coated with a ligand protein (orwith streptavidin followed by the binding of a biotinylated ligand) andblocked with the Blotto solution (Pierce). Purified phages (˜10¹⁰ pfu)originating from single plaques (M13)/colonies (fUSE5) are added to eachwell and incubated overnight at 4° C. After washing wells with anappropriate buffer (see above), bound phages are detected by thestandard ELISA protocol using anti-M13 Ab (rabbit, Sigma) andanti-rabbit Ig-peroxidase conjugate (Pierce) or using anti-M 13Ab-peroxidase conjugate (Pharmacia). Colormetric assays are performedusing TMB (3,3′,5,5′-tetramethylbenzidine, Pierce). The high affinity ofprotein G to immunoglobulins present a special problem; Abs cannot beused in detection. Therefore, to detect anti-protein G Monobodies,fusion phages are immobilized in wells and the binding is then measuredusing biotinylated protein G followed by the detection usingstreptavidin-peroxidase conjugate.

Production of Soluble Monobodies

After preliminary characterization of mutant Fn3s using phage ELISA,mutant genes are subcloned into the expression vector pEW1. Mutantproteins are produced as His.tag fusion proteins and purified, and theirconformation, stability and ligand affinity are characterized.

III. Increased Stability of Fn3 Scaffolds

The definition of “higher stability” of a protein is the ability of aprotein to retain its three-dimensional structure required for functionat a higher temperature (in the case of thermal denaturation), and inthe presence of a higher concentration of a denaturing chemical reagentsuch as guanidine hydrochloride. This type of “stability” is generallycalled “conformational stability.” It has been shown that conformationalstability is correlated with resistance against proteolytic degradation,i.e., breakdown of protein in the body (Kamtekar et al. 1993).

Improving the conformational stability is a major goal in proteinengineering. Here, mutations have been developed by the inventor thatenhance the stability of the fibronectin type III domain (Fn3). Theinventor has developed a technology in which Fn3 is used as a scaffoldto engineer artificial binding proteins (Koide et al., 1998). It hasbeen shown that many residues in the surface loop regions of Fn3 can bemutated without disrupting the overall structure of the Fn3 molecule,and that variants of Fn3 with a novel binding function can be engineeredusing combinatorial library screening (Koide et al., 1998). The inventorfound that, although Fn3 is an excellent scaffold, Fn3 variants thatcontain large number of mutations are destablized against chemicaldenaturation, compared to the wild-type'Fn3 protein (Koide et al.,1998). Thus, as the number of mutated positions are mutated in order toengineer a new binding function, the stability of such Fn3 variantsfurther decreases, ultimately leading to marginally stable proteins.Because artificial binding proteins must maintain theirthree-dimensional structure to be functional, stability limits thenumber of mutations that can be introduced in the scaffold. Thus,modifications of the Fn3 scaffold that increase its stability are usefulin that they allow one to introduce more mutations for better function,and that they make it possible to use Fn3-based engineered proteins in awider range of applications.

The inventor found that wild-type Fn3 is more stable at acidic pH thanat neutral pH (Koide et al., 1998). The pH dependence of Fn3 stabilityis characterized in FIG. 18. The pH dependence curve has an apparenttransition midpoint near pH 4 (FIG. 18). These results suggest that byidentifying and removing destablizing interactions in Fn3 one is able toimprove the stability of Fn3 at neutral pH. It should be noted that mostapplications of engineered Fn3, such as diagnostics, therapeutics andcatalysts, are expected to be used near neutral pH, and thus it isimportant to improve the stability at neutral pH. Studies by otherinvestigators have demonstrated that the optimization of surfaceelectrostatic properties can lead to a substantial increase in proteinstability (Peri et al. 2000, Spector et al. 1999, Loladze et al. 1999,Grimsley et al. 1999).

The pH dependence of Fn3 stability suggests that amino acids with pK_(a)near 4 are involved in the observed transition. The carboxyl groups ofaspartic acid (Asp) and glutamic acid (Glu) have pK_(a) in this range(Creighton, T. E. 1993). It is well known that if a carboxyl group hasunfavorable (i.e. destabilizing) interactions in a protein, its pK_(a)is shifted to a higher value from its standard, unperturbed value (Yangand Honig 1992). Thus, the pK_(a) values of all carboxyl groups in Fn3were determined using nuclear magnetic resonance (NMR) spectroscopy, toidentify carboxyl groups with unusual pK_(a)'s, as shown below.

First, the ¹³C resonance for the carboxyl carbon of each Asp and Gluresidue were assigned (FIG. 19). Next pH titration of ¹³C resonances wasperformed for these groups (FIG. 20). The pK_(a) values for theseresidues are listed in Table 3.

TABLE 3 pK_(a) values for Asp and Glu residues in Fn3. Residue pK_(a) E95.09 E38 3.79 E47 3.94 D3 3.66 D7 3.54, 5.54* D23 3.54, 5.25* D67 4.18D80 3.40 The standard deviation in the pK_(a) values are less than 0.05pH units. *Data for D7 and D23 were fitted with a transition curve withtwo pK_(a) values.These results show that Asp 7 and 23, and Glu 9 have up-shifted pK_(a)'swith respect to their unperturbed pK_(a)'s (approximately 4.0),indicating that these residues are involved in unfavorable interactions.In contrast, the other Asp and Glu residues have pK_(a)'s close to therespective unperturbed values, indicating that the carboxyl groups ofthese residues do not significantly contribute to the stability of Fn3.

In the three-dimensional structure of Fn3 (Main et al. 1992), Asp 7 and23, and Glu 9 form a patch on the surface (FIG. 21), with Asp 7centrally located in the patch. This spatial proximity of thesenegatively charged residues explains why these residues have unfavorableinteractions in Fn3. At low pH where these residues are protonated andneutral, the unfavorable interactions are expected to be mostlyrelieved. At the same time, the structure suggests that the stability ofFn3 at neutral pH could be improved if the electrostatic repulsionbetween these three residues is removed. Because Asp 7 is centrallylocated among the three residues, it was decided to mutate Asp 7. Twomutants were prepared, D7N and D7K (i.e., the aspartic acid at aminoacid residue number 7 was substituted with an asparagine residue or alysine residue, respectively). The former replaces the negative chargewith a neutral residue of virtually the same size. The latter places apositive charge at residue 7.

The degrees of stability of the mutant proteins were characterized inthermal and chemical denaturation measurements. In thermal denaturationmeasurements, denaturation of the Fn3 proteins was monitored usingcircular dichroism spectroscopy at the wavelength of 227 nm. All theproteins underwent a cooperative transition (FIG. 22). From thetransition curves, the midpoints of the transition (T_(m)) for thewild-type, D7N and D7K were determined to be 62, 69 and 70° C. in 0.02 Msodium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride and6.2 M urea. Thus, the mutations increased the T_(m) of wild-type Fn3 by7-8° C.

Chemical denaturation of Fn3 proteins was monitored using fluorescenceemission from the single Tip residue of Fn3 (FIG. 23). The free energiesof unfolding in the absence of guanidine HCl (ΔG⁰) were determined to be7.4, 8.1 and 8.0 kcal/mol for the wild-type, D7N and D7K, respectively(a larger ΔG⁰ indicates a higher stability). The two mutants were againfound to be more stable than the wild-type protein.

These results show that a point mutation on the surface cansignificantly enhance the stability of Fn3. Because these mutations areon the surface, they minimally alter the structure of Fn3, and they canbe easily introduced to other, engineered Fn3 proteins. In addition,mutations at Glu 9 and/or Asp 23 also enhance the stability of Fn3.Furthermore, mutations at one or more of these three residues can becombined.

Thus, Fn3 is the fourth example of a monomeric immunoglobulin-likescaffold that can be used for engineering binding proteins. Successfulselection of novel binding proteins have also been based on minibody,tendamistat and “camelized” immunoglobulin VH domain scaffolds (Martinet al., 1994; Davies & Riechmann, 1995; McConnell & Hoess, 1995). TheFn3 scaffold has advantages over these systems. Bianchi et al. reportedthat the stability of a minibody was 2.5 kcal/mol, significantly lowerthan that of Ubi4-K. No detailed structural characterization ofminibodies has been reported to date. Tendamistat and the VH domaincontain disulfide bonds, and thus preparation of correctly foldedproteins may be difficult. Davies and Riechmann reported that the yieldsof their camelized VH domains were less than 1 mg per liter culture(Davies & Riechmann, 1996).

Thus, the Fn3 framework can be used as a scaffold for molecularrecognition. Its small size, stability and well-characterized structuremake Fn3 an attractive system. In light of the ubiquitous presence ofFn3 in a wide variety of natural proteins involved in ligand binding,one can engineer Fn3-based binding proteins to different classes oftargets.

The following examples are intended to illustrate but not limit theinvention.

Example I Construction of the Fn3 Gene

A synthetic gene for tenth Fn3 of fibronectin (FIG. 1) was designed onthe basis of amino acid residue 1416-1509 of human fibronectin(Kornblihtt, et al., 1985) and its three dimensional structure (Main, etal., 1992). The gene was engineered to include convenient restrictionsites for mutagenesis and the so-called “preferred codons” for highlevel protein expression (Gribskov, et al., 1984) were used. Inaddition, a glutamine residue was inserted after the N-terminalmethionine in order to avoid partial processing of the N-terminalmethionine which often degrades NMR spectra (Smith, et al., 1994).Chemical reagents were of the analytical grade or better and purchasedfrom Sigma Chemical Company and J. T. Baker, unless otherwise noted.Recombinant DNA procedures were performed as described in “MolecularCloning” (Sambrook, et al., 1989), unless otherwise stated. Customoligonucleotides were purchased from Operon Technologies. Restrictionand modification enzymes were from New England Biolabs.

The gene was assembled in the following manner. First, the gene sequence(FIG. 5) was divided into five parts with boundaries at designedrestriction sites: fragment 1, NdeI-PstI (oligonucleotides FN1F and FN1R(Table 2); fragment 2, PstI-EcoRI (FN2F and FN2R); fragment 3,EcoRI-SalI (FN3F and FN3R); fragment 4, SalI-SacI (FN4F and FN4R);fragment 5, SacI-BamHI (FN5F and FN5R). Second, for each part, a pair ofoligonucleotides which code opposite strands and have complementaryoverlaps of approximately 15 bases was synthesized. Theseoligonucleotides were designated FN1F-FN5R and are shown in Table 2.Third, each pair (e.g., FN1F and FN1R) was annealed and single-strandregions were filled in using the Klenow fragment of DNA polymerase.Fourth, the double stranded oligonucleotide was digested with therelevant restriction enzymes at the termini of the fragment and clonedinto the pBlueScript SK plasmid (Stratagene) which had been digestedwith the same enzymes as those used for the fragments. The DNA sequenceof the inserted fragment was confirmed by DNA sequencing using anApplied Biosystems DNA sequencer and the dideoxy termination protocolprovided by the manufacturer. Last, steps 2-4 were repeated to obtainthe entire gene.

The gene was also cloned into the pET3a and pET15b (Novagen) vectors(pAS45 and pAS25, respectively). The maps of the plasmids are shown inFIGS. 6 and 7. E. coli BL21 (DE3) (Novagen) containing these vectorsexpressed the Fn3 gene under the control of bacteriophage T7 promotor(Studier, et al., 1990); pAS24 expresses the 96-residue Fn3 proteinonly, while pAS45 expresses Fn3 as a fusion protein with poly-histidinepeptide (His.tag). High level expression of the Fn3 protein and itsderivatives in E. coli was detected as an intense band on SDS-PAGEstained with CBB.

The binding reaction of the monobodies is characterized quantitativelyby means of fluorescence spectroscopy using purified soluble monobodies.

Intrinsic fluorescence is monitored to measure binding reactions. Trpfluorescence (excitation at ˜290 nm, emission at 300 350 nm) and Tyrfluorescence (excitation at ˜260 nm, emission at ˜303 nm) is monitoredas the Fn3-mutant solution (≦100 μM) is titrated with a ligand solution.When a ligand is fluorescent (e.g. fluorescein), fluorescence from theligand may be used. K_(d) of the reaction will be determined by thenonlinear least-squares fitting of the bimolecular binding equation.

If intrinsic fluorescence cannot be used to monitor the bindingreaction, monobodies are labeled with fluorescein-NHS (Pierce) andfluorescence polarization is used to monitor the binding reaction (Burkeet al., 1996).

Example II Modifications to Include Restriction Sites in the Fn3 Gene

The restriction sites were incorporated in the synthetic Fn3 genewithout changing the amino acid sequence Fn3. The positions of therestriction sites were chosen so that the gene construction could becompleted without synthesizing long (>60 bases) oligonucleotides and sothat two loop regions could be mutated (including by randomization) bythe cassette mutagenesis method (i.e., swapping a fragment with anothersynthetic fragment containing mutations). In addition, the restrictionsites were chosen so that most sites were unique in the vector for phagedisplay. Unique restriction sites allow one to recombine monobody cloneswhich have been already selected in order to supply a larger sequencespace.

Example III Construction of M13 Phage Display Libraries

A vector for phage display, pAS38 (for its map, see FIG. 8) wasconstructed as follows. The XbaI-BamHI fragment of pET12a encoding thesignal peptide of OmpT was cloned at the 5′ end of the Fn3 gene. TheC-terminal region (from the FN5F and FN5R oligonucleotides, see Table 2)of the Fn3 gene was replaced with a new fragment consisting of the FN5Fand FN5R′ oligonucleotides (Table 2) which introduced a MluI site and alinker sequence for making a fusion protein with the pIII protein ofbacteriophage M13. A gene fragment coding the C-terminal domain of M13pIII was prepared from the wild-type gene III of M13 mp18 using PCR(Corey, et al., 1993) and the fragment was inserted at the 3′ end of theOmpT-Fn3 fusion gene using the MluI and HindIII sites.

Phages were produced and purified using a helper phage, M13K07,according to a standard method (Sambrook, et al., 1989) except thatphage particles were purified by a second polyethylene glycolprecipitation. Successful display of Fn3 on fusion phages was confirmedby ELISA (Harlow & Lane, 1988) using an antibody against fibronectin(Sigma) and a custom anti-FN3 antibody (Cocalico Biologicals, PA, USA).

Example IV Libraries Containing Loop Variegations in the AB Loop

A nucleic acid phage display library having variegation in the AB loopis prepared by the following methods. Randomization is achieved by theuse of oligonucleotides containing degenerated nucleotide sequence.Residues to be variegated are identified by examining the X-ray and NMRstructures of Fn3 (Protein Data Bank accession numbers, 1FNA and 1TTF,respectively). Oligonucleotides containing NNK (N and K here denote anequimolar mixture of A, T, G, and C and an equimolar mixture of G and T,respectively) for the variegated residues are synthesized (seeoligonucleotides BC3, FG2, FG3, and FG4 in Table 2 for example). The NNKmixture codes for all twenty amino acids and one termination codon(TAG). TAG, however, is suppressed in the E. coli XL-1 blue.Single-stranded DNAs of pAS38 (and its derivatives) are prepared using astandard protocol (Sambrook, et al., 1989).

Site-directed mutagenesis is performed following published methods (seefor example, Kunkel, 1985) using a Muta-Gene kit (BioRad). The librariesare constructed by electroporation of E. coli XL-1 Blue electroporationcompetent cells (200 μl; Stratagene) with 1 μg of the plasmid DNA usinga BTX electrocell manipulator ECM 395 1 mm gap cuvette. A portion of thetransformed cells is plated on an LB-agar plate containing ampicillin(100 μg/ml) to determine the transformation efficiency. Typically, 3×10transformants are obtained with 1 μg of DNA, and thus a library contains10⁸ to 10⁹ independent clones. Phagemid particles were prepared asdescribed above.

Example V Loop Variegations in the BC, CD, DE, EF or FG Loop

A nucleic acid phage display library having five variegated residues(residues number 26-30) in the BC loop, and one having seven variegatedresidues (residue numbers 78-84) in the FG loop, was prepared using themethods described in Example IV above. Other nucleic acid phage displaylibraries having variegation in the CD, DE or EF loop can be prepared bysimilar methods.

Example VI Loop Variegations in the FG and BC Loop

A nucleic acid phage display library having seven variegated residues(residues number 78-84) in the FG loop and five variegated residues(residue number 26-30) in the BC loop was prepared. Variegations in theBC loop were prepared by site-directed mutagenesis (Kunkel, et al.)using the BC3 oligonucleotide described in Table 1. Variegations in theFG loop were introduced using site-directed mutagenesis using the BCloop library as the starting material, thereby resulting in librariescontaining variegations in both BC and FG loops. The oligonucleotide FG2has variegating residues 78-84 and oligonucleotide FG4 has variegatingresidues 77-81 and a deletion of residues 82-84.

A nucleic acid phage display library having five variegated residues(residues 78-84) in the FG loop and a three residue deletion (residues82-84) in the FG loop, and five variegated residues (residues 26-30) inthe BC loop, was prepared. The shorter FG loop was made in an attempt toreduce the flexibility of the FG loop; the loop was shown to be highlyflexible in Fn3 by the NMR studies of Main, et al. (1992). A highlyflexible loop may be disadvantageous to forming a binding site with ahigh affinity (a large entropy loss is expected upon the ligand binding,because the flexible loop should become more rigid). In addition, otherFn3 domains (besides human) have shorter FG loops (for sequencealignment, see FIG. 12 in Dickinson, et al. (1994)).

Randomization was achieved by the use of oligonucleotides containingdegenerate nucleotide sequence (oligonucleotide BC3 for variegating theBC loop and oligonucleotides FG2 and FG4 for variegating the FG loops).

Site-directed mutagenesis was performed following published methods (seefor example, Kunkel, 1985). The libraries were constructed byelectrotransforming E. coli XL-1 Blue (Stratagene). Typically a librarycontains 10⁸ to 10⁹ independent clones. Library 2 contains fivevariegated residues in the BC loop and seven variegated residues in theFG loop. Library 4 contains five variegated residues in each of the BCand FG loops, and the length of the FG loop was shortened by threeresidues.

Example VII fd Phage Display Libraries Constructed with LoopVariegations

Phage display libraries are constructed using the fd phage as thegenetic vector. The Fn3 gene is inserted in fUSE5 (Parmley & Smith,1988) using SfiI restriction sites which are introduced at the 5′ and 3′ends of the Fn3 gene using PCR. The expression of this phage results inthe display of the fusion pIII protein on the surface of the fd phage.Variegations in the Fn3 loops are introduced using site-directedmutagenesis as described hereinabove, or by subcloning the Fn3 librariesconstructed in M13 phage into the fUSE5 vector.

Example VIII Other Phage Display Libraries

T7 phage libraries (Novagen, Madison, Wis.) and bacterial piliexpression systems (Invitrogen) are also useful to express the Fn3 gene.

Example IX Isolation of Polypeptides which Bind to MacromolecularStructures

The selection of phage-displayed monobodies was performed following theprotocols of Barbas and coworkers (Rosenblum & Barbas, 1995). Briefly,approximately 1 μg of a target molecule (“antigen”) in sodium carbonatebuffer (100 mM, pH 8.5) was immobilized in the wells of a microtiterplate (Maxisorp, Nunc) by incubating overnight at 4° C. in an air tightcontainer. After the removal of this solution, the wells were thenblocked with a 3% solution of BSA (Sigma, Fraction V) in TBS byincubating the plate at 37° C. for 1 hour. A phagemid library solution(50 μl) containing approximately 10¹² colony forming units (cfu) ofphagemid was absorbed in each well at 37° C. for 1 hour. The wells werethen washed with an appropriate buffer (typically TBST, 50 mM Tris-HCl(pH 7.5), 150 mM NaCl, and 0.5% Tween20) three times (once for the firstround). Bound phage were eluted by an acidic solution (typically, 0.1 Mglycine-HCl, pH 2.2; 50 μl) and recovered phage were immediatelyneutralized with 3 μl of Tris solution. Alternatively, bound phage wereeluted by incubating the wells with 50 μl of TBS containing the antigen(1-10 μM). Recovered phage were amplified using the standard protocolemploying the XL1Blue cells as the host (Sambrook, et al.). Theselection process was repeated 5-6 times to concentrate positive clones.After the final round, individual clones were picked and their bindingaffinities and DNA sequences were determined.

The binding affinities of monobodies on the phage surface werecharacterized using the phage ELISA technique (Li, et al., 1995). Wellsof microtiter plates (Nunc) were coated with an antigen and blocked withBSA. Purified phages (10⁸-10¹¹ cfu) originating from a single colonywere added to each well and incubated 2 hours at 37° C. After washingwells with an appropriate buffer (see above), bound phage were detectedby the standard ELISA protocol using anti-M13 antibody (rabbit, Sigma)and anti-rabbit Ig-peroxidase conjugate (Pierce). Colorimetric assayswere performed using Turbo-TMB (3,3′,5,5′-tetramethylbenzidine, Pierce)as a substrate.

The binding affinities of monobodies on the phage surface were furthercharacterized using the competition ELISA method (Djavadi-Ohaniance, etal., 1996). In this experiment, phage ELISA is performed in the samemanner as described above, except that the phage solution contains aligand at varied concentrations. The phage solution was incubated a 4°C. for one hour prior to the binding of an immobilized ligand in amicrotiter plate well. The affinities of phage displayed monobodies areestimated by the decrease in ELISA signal as the free ligandconcentration is increased.

After preliminary characterization of monobodies displayed on thesurface of phage using phage ELISA, genes for positive clones weresubcloned into the expression vector pAS45. E. coli BL21(DE3) (Novagen)was transformed with an expression vector (pAS45 and its derivatives).Cells were grown in M9 minimal medium and M9 medium supplemented withBactotryptone (Difco) containing ampicillin (200 μg/ml). For isotopiclabeling, ¹⁵N NH₄Cl and/or ¹³C glucose replaced unlabeled components.Stable isotopes were purchased from Isotec and Cambridge Isotope Labs.500 ml medium in a 2 1 baffle flask was inoculated with 10 ml ofovernight culture and agitated at approximately 140 rpm at 37° C. IPTGwas added at a final concentration of 1 mM to induce protein expressionwhen OD(600 nm) reached approximately 1.0. The cells were harvested bycentrifugation 3 hours after the addition of IPTG and kept frozen at−70° C. until used.

Fn3 and monobodies with His.tag were purified as follows. Cells weresuspended in 5 ml/(g cell) of 50 mM Tris (pH 7.6) containing 1 mMphenylmethylsulfonyl fluoride. HEL (Sigma, 3× crystallized) was added toa final concentration of 0.5 mg/ml. After incubating the solution for 30min at 37° C., it was sonicated so as to cause cell breakage three timesfor 30 seconds on ice. Cell debris was removed by centrifugation at15,000 rpm in an Sorval RC-2B centrifuge using an SS-34 rotor.Concentrated sodium chloride is added to the solution to a finalconcentration of 0.5 M. The solution was then applied to a 1 ml HisTrap™chelating column (Pharmacia) preloaded with nickel chloride (0.1 M, 1ml) and equilibrated in the Tris buffer (50 mM, pH 8.0) containing 0.5 Msodium chloride. After washing the column with the buffer, the boundprotein was eluted with a Tris buffer (50 mM, pH 8.0) containing 0.5 Mimidazole. The His.tag portion was cleaved off, when required, bytreating the fusion protein with thrombin using the protocol supplied byNovagen (Madison, Wis.). Fn3 was separated from the His.tag peptide andthrombin by a Resources®column (Pharmacia) using a linear gradient ofsodium chloride (0-0.5 M) in sodium acetate buffer (20 mM, pH 5.0).

Small amounts of soluble monobodies were prepared as follows. XL-1 Bluecells containing pAS38 derivatives (plasmids coding Fn3-pIII fusionproteins) were grown in LB media at 37° C. with vigorous shaking untilOD(600 nm) reached approximately 1.0; IPTG was added to the culture to afinal concentration of 1 mM, and the cells were further grown overnightat 37° C. Cells were removed from the medium by centrifugation, and thesupernatant was applied to a microtiter well coated with a ligand.Although XL-1 Blue cells containing pAS38 and its derivatives expressFN3-pIII fusion proteins, soluble proteins are also produced due to thecleavage of the linker between the Fn3 and pIII regions by proteolyticactivities of E. coli (Rosenblum & Barbas, 1995). Binding of a monobodyto the ligand was examined by the standard ELISA protocol using a customantibody against Fn3 (purchased from Cocalico Biologicals, Reamstown,Pa.). Soluble monobodies obtained from the periplasmic fraction of E.coli cells using a standard osmotic shock method were also used.

Example X Ubiquitin Binding Monobody

Ubiquitin is a small (76 residue) protein involved in the degradationpathway in eukaryotes. It is a single domain globular protein. Yeastubiquitin was purchased from Sigma Chemical Company and was used withoutfurther purification.

Libraries 2 and 4, described in Example VI above, were used to selectubiquitin-binding monobodies. Ubiquitin (1 μg in 50 μl sodiumbicarbonate buffer (100 mM, pH 8.5)) was immobilized in the wells of amicrotiter plate, followed by blocking with BSA (3% in TBS). Panning wasperformed as described above. In the first two rounds, 1 μg of ubiquitinwas immobilized per well, and bound phage were elute with an acidicsolution. From the third to the sixth rounds, 0.1 μg of ubiquitin wasimmobilized per well and the phage were eluted either with an acidicsolution or with TBS containing 10 μM ubiquitin.

Binding of selected clones was tested first in the polyclonal mode,i.e., before isolating individual clones. Selected clones from alllibraries showed significant binding to ubiquitin. These results areshown in FIG. 9. The binding to the immobilized ubiquitin of the cloneswas inhibited almost completely by less than 30 μM soluble ubiquitin inthe competition ELISA experiments (see FIG. 10). The sequences of the BCand FG loops of ubiquitin-binding monobodies is shown in Table 4.

TABLE 4 Sequences of ubiquitin-binding monobodies Occurrence (if moreName BC loop FG loop than one) 211 CARRA RWIPLAK 2 (SEQ ID NO: 31)(SEQ ID NO: 32) 212 CWRRA RWVGLAW (SEQ ID NO: 33) (SEQ ID NO: 34) 213CKHRR FADLWWR (SEQ ID NO: 35) (SEQ ID NO: 36) 214 CRRGR RGFMWLS(SEQ ID NO: 37) (SEQ ID NO: 38) 215 CNWRR RAYRYRW (SEQ ID NO: 39)(SEQ ID NO: 40) 411 SRLRR PPWRV 9 (SEQ ID NO: 41) (SEQ ID NO: 42) 422ARWTL RRWWW (SEQ ID NO: 43) (SEQ ID NO: 44) 424 GQRTF RRWWA(SEQ ID NO: 45) (SEQ ID NO: 46)

The 411 clone, which was the most enriched clone, was characterizedusing phage ELISA. The 411 clone showed selective binding and inhibitionof binding in the presence of about 10 μM ubiquitin in solution (FIG.11).

Example XI Methods for the Immobilization of Small Molecules

Target molecules were immobilized in wells of a microtiter plate(Maxisorp, Nunc) as described hereinbelow, and the wells were blockedwith BSA. In addition to the use of carrier protein as described below,a conjugate of a target molecule in biotin can be made. The biotinylatedligand can then be immobilized to a microtiter plate well which has beencoated with streptavidin.

In addition to the use of a carrier protein as described below, onecould make a conjugate of a target molecule and biotin (Pierce) andimmobilize a biotinylated ligand to a microtiter plate well which hasbeen coated with streptavidin (Smith and Scott, 1993).

Small molecules may be conjugated with a carrier protein such as bovineserum albumin (BSA, Sigma), and passively adsorbed to the microtiterplate well. Alternatively, methods of chemical conjugation can also beused. In addition, solid supports other than microtiter plates canreadily be employed.

Example XII Fluorescein Binding Monobody

Fluorescein has been used as a target for the selection of antibodiesfrom combinatorial libraries (Barbas, et al. 1992). NHS-fluorescein wasobtained from Pierce and used according to the manufacturer'sinstructions in preparing conjugates with BSA (Sigma). Two types offluorescein-BSA conjugates were prepared with approximate molar ratiosof 17 (fluorescein) to one (BSA).

The selection process was repeated 5-6 times to concentrate positiveclones. In this experiment, the phage library was incubated with aprotein mixture (BSA, cytochrome C (Sigma, Horse) and RNaseA (Sigma,Bovine), 1 mg/ml each) at room temperature for 30 minutes, prior to theaddition to ligand coated wells. Bound phage were eluted in TBScontaining 10 μM soluble fluorescein, instead of acid elution. After thefinal round, individual clones were picked and their binding affinities(see below) and DNA sequences were determined.

TABLE 5 BC FG Clones from Library #2 WT AVTVR RGDSPAS (SEQ ID NO: 47)(SEQ ID NO: 48) pLB24.1 CNWRR RAYRYRW (SEQ ID NO: 49) (SEQ ID NO: 50)pLB24.2 CMWRA RWGMLRR (SEQ ID NO: 51) (SEQ ID NO: 52) pLB24.3 ARMRERWLRGRY (SEQ ID NO: 53) (SEQ ID NO: 54) pLB24.4 CARRR RRAGWGW(SEQ ID NO: 55) (SEQ ID NO: 56) pLB24.5 CNWRR RAYRYRW (SEQ ID NO: 57)(SEQ ID NO: 58) pLB24.6 RWRER RHPWTER (SEQ ID NO: 59) (SEQ ID NO: 60)pLB24.7 CNWRR RAYRYRW (SEQ ID NO: 61) (SEQ ID NO: 62) pLB24.8 ERRVPRLLLWQR (SEQ ID NO: 63) (SEQ ID NO: 64) pLB24.9 GRGAG FGSFERR(SEQ ID NO: 65) (SEQ ID NO: 66) pLB24.11  CRWTR RRWFDGA (SEQ ID NO: 67)(SEQ ID NO: 68) pLB24.12  CNWRR RAYRYRW (SEQ ID NO: 69) (SEQ ID NO: 70)Clones from Library #4 WT AVTVR GRGDS (SEQ ID NO: 71) (SEQ ID NO: 72)pLB25.1 GQRTF RRWWA (SEQ ID NO: 73) (SEQ ID NO: 74) pLB25.2 GQRTF RRWWA(SEQ ID NO: 75) (SEQ ID NO: 76) pLB25.3 GQRTF RRWWA (SEQ ID NO: 77)(SEQ ID NO: 78) pLB25.4 LRYRS GWRWR (SEQ ID NO: 79) (SEQ ID NO: 80)pLB25.5 GQRTF RRWWA (SEQ ID NO: 81) (SEQ ID NO: 82) pLB25.6 GQRTF RRWWA(SEQ ID NO: 83) (SEQ ID NO: 84) pLB25.7 LRYRS GWRWR (SEQ ID NO: 85)(SEQ ID NO: 86) pLB25.9 LRYRS GWRWR (SEQ ID NO: 87) (SEQ ID NO: 88)pLB25.11  GQRTF RRWWA (SEQ ID NO: 89) (SEQ ID NO: 90) pLB25.12  LRYRSGWRWR (SEQ ID NO: 91) (SEQ ID NO: 92)

Preliminary characterization of the binding affinities of selectedclones were performed using phage ELISA and competition phage ELISA (seeFIG. 12 (Fluorescein-1) and FIG. 13 (Fluorescein-2)). The four clonestested showed specific binding to the ligand-coated wells, and thebinding reactions are inhibited by soluble fluorescein (see FIG. 13).

Example XIII Digoxigenin Binding Monobody

Digoxigenin-3-O-methyl-carbonyl-e-aminocapronic acid-NHS (BoehringerMannheim) is used to prepare a digoxigenin-BSA conjugate. The couplingreaction is performed following the manufacturers' instructions. Thedigoxigenin-BSA conjugate is immobilized in the wells of a microtiterplate and used for panning. Panning is repeated 5 to 6 times to enrichbinding clones. Because digoxigenin is sparingly soluble in aqueoussolution, bound phages are eluted from the well using acidic solution.See Example XIV.

Example XIV TSAC (Transition State Analog Compound) Binding Monobodies

Carbonate hydrolyzing monobodies are selected as follows. A transitionstate analog for carbonate hydrolysis, 4-nitrophenyl phosphonate issynthesized by an Arbuzov reaction as described previously (Jacobs andSchultz, 1987). The phosphonate is then coupled to the carrier protein,BSA, using carbodiimide, followed by exhaustive dialysis (Jacobs andSchultz, 1987). The hapten-BSA conjugate is immobilized in the wells ofa microtiter plate and monobody selection is performed as describedabove. Catalytic activities of selected monobodies are tested using4-nitrophenyl carbonate as the substrate.

Other haptens useful to produce catalytic monobodies are summarized inH. Suzuki (1994) and in N. R. Thomas (1994).

Example XV NMR Characterization of Fn3 and Comparison of the Fn3Secreted by Yeast with that Secreted by E. coli

Nuclear magnetic resonance (NMR) experiments are performed to identifythe contact surface between FnAb and a target molecule, e.g., monobodiesto fluorescein, ubiquitin, RNaseA and soluble derivatives ofdigoxigenin. The information is then be used to improve the affinity andspecificity of the monobody. Purified monobody samples are dissolved inan appropriate buffer for NMR spectroscopy using Amicon ultrafiltrationcell with a YM-3 membrane. Buffers are made with 90% H₂O/10% D₂O(distilled grade, Isotec) or with 100% D₂O. Deuterated compounds (e.g.acetate) are used to eliminate strong signals from them.

NMR experiments are performed on a Varian Unity INOVA 600 spectrometerequipped with four RF channels and a triple resonance probe with pulsedfield gradient capability. NMR spectra are analyzed using processingprograms such as Felix (Molecular Simulations), nmrPipe, PIPP, and CAPP(Garrett, et al., 1991; Delaglio, et al., 1995) on UNIX workstations.Sequence specific resonance assignments are made using well-establishedstrategy using a set of triple resonance experiments (CBCA(CO)NH andHNCACB) (Grzesiek & Bax, 1992; Wittenkind & Mueller, 1993).

Nuclear Overhauser effect (NOE) is observed between ¹H nuclei closerthan approximately 5 Å, which allows one to obtain information oninterproton distances. A series of double- and triple-resonanceexperiments (Table 6; for recent reviews on these techniques, see Bax &Grzesiek, 1993 and Kay, 1995) are performed to collect distance (i.e.NOE) and dihedral angle (J-coupling) constraints. Isotope-filteredexperiments are performed to determine resonance assignments of thebound ligand and to obtain distance constraints within the ligand andthose between FnAb and the ligand. Details of sequence specificresonance assignments and NOE peak assignments have been described indetail elsewhere (Clore & Gronenborn, 1991; Pascal, et al., 1994b;Metzler, et al., 1996).

TABLE 6 NMR experiments for structure characterization Experiment NameReference 1. reference spectra 2D-¹H, ¹⁵N-HSQC (Bodenhausen & Ruben,1980; Kay, et al., 1992) 2D-¹H, ¹³C-HSQC (Bodenhausen & Ruben, 1980;Vuister & Bax, 1992) 2. backbone and side chain resonance assignments of¹³C/¹⁵N- labeled protein 3D-CBCA(CO)NH (Grzesiek & Bax, 1992) 3D-HNCACB(Wittenkind & Mueller, 1993) 3D-C(CO)NH (Logan et al., 1992; Grzesiek etal., 1993) 3D-H(CCO)NH 3D-HBHA(CBCACO)NH (Grzesiek & Bax, 1993)3D-HCCH-TOCSY (Kay et al., 1993) 3D-HCCH-COSY (Ikura et al., 1991)3D-¹H, ¹⁵N-TOCSY-HSQC (Zhang et al., 1994) 2D-HB(CBCDCE)HE (Yamazaki etal., 1993) 3. resonance assignments of unlabeled ligand2D-isotope-filtered ¹H-TOCSY 2D-isotope-filtered ¹H-COSY2D-isotope-filtered ¹H-NOESY (Ikura & Bax, 1992) 4. structuralconstraints within labeled protein 3D-¹H, ¹⁵N-NOESY-HSQC (Zhang et al.,1994) 4D-¹H, ¹³C-HMQC-NOESY-HMQC (Vuister et al., 1993) 4D-¹H, ¹³C,¹⁵N-HSQC-NOESY-HSQC (Muhandiram et al., within unlabeled ligand 1993;Pascal et al., 1994a) 2D-isotope-filtered ¹H-NOESY (Ikura & Bax, 1992)interactions between protein and ligand 3D-isotope-filtered ¹H,¹⁵N-NOESY- HSQC 3D-isotope-filtered ¹H, ¹³C-NOESY- (Lee et al., 1994)HSQC 5. dihedral angle constraints J-molulated ¹H, ¹⁵N-HSQC (Billeter etal., 1992) 3D-HNHB (Archer et al., 1991)

Backbone ¹H, ¹⁵N and ¹³C resonance assignments for a monobody arecompared to those for wild-type Fn3 to assess structural changes in themutant. Once these data establish that the mutant retains the globalstructure, structural refinement is performed using experimental NOEdata. Because the structural difference of a monobody is expected to beminor, the wild-type structure can be used as the initial model aftermodifying the amino acid sequence. The mutations are introduced to thewild-type structure by interactive molecular modeling, and then thestructure is energy-minimized using a molecular modeling program such asQuanta (Molecular Simulations). Solution structure is refined usingcycles of dynamical simulated annealing (Nilges et al., 1988) in theprogram X-PLOR (Brünger, 1992). Typically, an ensemble of fiftystructures is calculated. The validity of the refined structures isconfirmed by calculating a fewer number of structures from randomlygenerated initial structures in X-PLOR using the YASAP protocol (Nilges,et al., 1991). Structure of a monobody-ligand complex is calculated byfirst refining both components individually using intramolecular NOEs,and then docking the two using intermolecular NOEs.

For example, the ¹H, ¹⁵N-HSQC spectrum for the fluorescein-bindingmonobody LB25.5 is shown in FIG. 14. The spectrum shows a gooddispersion (peaks are spread out) indicating that LB25.5 is folded intoa globular conformation. Further, the spectrum resembles that for thewild-type Fn3, showing that the overall structure of LB25.5 is similarto that of Fn3. These results demonstrate that ligand-binding monobodiescan be obtained without changing the global fold of the Fn3 scaffold.

Chemical shift perturbation experiments are performed by forming thecomplex between an isotope-labeled FnAb and an unlabeled ligand. Theformation of a stoichiometric complex is followed by recording the HSQCspectrum. Because chemical shift is extremely sensitive to nuclearenvironment, formation of a complex usually results in substantialchemical shift changes for resonances of amino acid residues in theinterface. Isotope-edited NMR experiments (2D HSQC and 3D CBCA(CO)NH)are used to identify the resonances that are perturbed in the labeledcomponent of the complex; i.e. the monobody. Although the possibility ofartifacts due to long-range conformational changes must always beconsidered, substantial differences for residues clustered on continuoussurfaces are most likely to arise from direct contacts (Chen et al.,1993; Gronenborn & Clore, 1993).

An alternative method for mapping the interaction surface utilizes amidehydrogen exchange (HX) measurements. HX rates for each amide proton aremeasured for ¹⁵N labeled monobody both, free and complexed with aligand. Ligand binding is expected to result in decreased amide HX ratesfor monobody residues in the interface between the two proteins, thusidentifying the binding surface. HX rates for monobodies in the complexare measured by allowing HX to occur for a variable time followingtransfer of the complex to D₂O; the complex is dissociated by loweringpH and the HSQC spectrum is recorded at low pH where amide HX is slow.Fn3 is stable and soluble at low pH, satisfying the prerequisite for theexperiments.

Example XVI Construction and Analysis of Fn3-Display System Specific forUbiquitin

An Fn3-display system was designed and synthesized, ubiquitin-bindingclones were isolated and a major Fn3 mutant in these clones wasbiophysically characterized.

Gene construction and phage display of Fn3 was performed as in ExamplesI and II above. The Fn3-phage pIII fusion protein was expressed from aphagemid-display vector, while the other components of the M13 phage,including the wild-type pIII, were produced using a helper phage (Basset al., 1990). Thus, a phage produced by this system should contain lessthan one copy of Fn3 displayed on the surface. The surface display ofFn3 on the phage was detected by ELISA using an anti-Fn3 antibody. Onlyphages containing the Fn3-pIII fusion vector reacted with the antibody.

After confirming the phage surface to display Fn3, a phage displaylibrary of Fn3 was constructed as in Example III. Random sequences wereintroduced in the BC and FG loops. In the first library, five residues(77-81) were randomized and three residues (S2-84) were deleted from theFG loop. The deletion was intended to reduce the flexibility and improvethe binding affinity of the FG loop. Five residues (26-30) were alsorandomized in the BC loop in order to provide a larger contact surfacewith the target molecule. Thus, the resulting library contains fiverandomized residues in each of the BC and FG loops (Table 7). Thislibrary contained approximately 10⁸ independent clones.

Library Screening

Library screening was performed using ubiquitin as the target molecule.In each round of panning, Fn3-phages were absorbed to a ubiquitin-coatedsurface, and bound phages were eluted competitively with solubleubiquitin. The recovery ratio improved from 4.3×10″⁷ in the second roundto 4.5×10⁻⁶ in the fifth round, suggesting an enrichment of bindingclones. After five founds of panning, the amino acid sequences ofindividual clones were determined (Table 7).

TABLE 7 Sequences in the variegated loops of enriched clones NameBC loop FG loop Frequency Wild GCAGTTACCGTGCGT GGCCGTGGTGACAGCCCAGCGAGC— Type (SEQ ID NO: 93) (SEQ ID NO: 95) AlaValThrValArgGlyArgGlyAspSerProAlaSer (SEQ ID NO: 94) (SEQ ID NO: 96) Library^(a)NNKNNKNNKNNKNNK NNKNNKNNKNNKNNK--------- — X X X X XX X X X X (deletion) clone1 TCGAGGTTGCGGCGG CCGCCGTGGAGGGTG 9 (Ubi4)(SEQ ID NO: 97) (SEQ ID NO: 99) SerArgLeuArgArg ProProTrpArgVal(SEQ ID NO: 98) (SEQ ID NO: 100) clone2 GGTCAGCGAACTTTT AGGCGGTGGTGGGCT1 (SEQ ID NO: 101) (SEQ ID NO: 103) GlyGlnArgThrPhe ArgArgTrpTrpAla(SEQ ID NO: 102) (SEQ ID NO: 104) clone3 GCGAGGTGGACGCTT AGGCGGTGGTGGTGG1 (SEQ ID NO: 105) (SEQ ID NO: 107) AlaArgTrpThrLeu ArgArgTrpTrpTrp(SEQ ID NO: 106) (SEQ ID NO: 108) ^(a)N denotes an equimolar mixture ofA, T, G and C; K denotes an equimolar mixture of G and T.

A clone, dubbed Ubi4, dominated the enriched pool of Fn3 variants.Therefore, further investigation was focused on this Ubi4 clone. Ubi4contains four mutations in the BC loop (Arg 30 in the BC loop wasconserved) and five mutations and three deletions in the FG loop. Thus13% (12 out of 94) of the residues were altered in Ubi4 from thewild-type sequence.

FIG. 15 shows a phage ELISA analysis of Ubi4. The Ubi4 phage binds tothe target molecule, ubiquitin, with a significant affinity, while aphage displaying the wild-type Fn3 domain or a phase with no displayedmolecules show little detectable binding to ubiquitin (FIG. 15 a). Inaddition, the Ubi4 phage showed a somewhat elevated level of backgroundbinding to the control surface lacking the ubiquitin coating. Acompetition ELISA experiments shows the IC₅₀ (concentration of the freeligand which causes 50% inhibition of binding) of the binding reactionis approximately 5 (FIG. 15 b). BSA, bovine ribonuclease A andcytochrome C show little inhibition of the Ubi4-ubiquitin bindingreaction (FIG. 15 c), indicating that the binding reaction of Ubi4 toubiquitin does result from specific binding.

Characterization of a Mutant Fn3 Protein

The expression system yielded 50-100 mg Fn3 protein per liter culture. Asimilar level of protein expression was observed for the Ubi4 clone andother mutant Fn3 proteins.

Ubi4-Fn3 was expressed as an independent protein. Though a majority ofUbi4 was expressed in E. coli as a soluble protein, its solubility wasfound to be significantly reduced as compared to that of wild-type Fn3.Ubi4 was soluble up to ˜20 μM at low pH, with much lower solubility atneutral pH. This solubility was not high enough for detailed structuralcharacterization using

NMR Spectroscopy or X-Ray Crystallography.

The solubility of the Ubi4 protein was improved by adding a solubilitytail, GKKGK (SEQ ID NO:109), as a C-terminal extension. The gene forUbi4-Fn3 was subcloned into the expression vector pAS45 using PCR. TheC-terminal solubilization tag, GKKGK (SEQ ID NO:109), was incorporatedin this step. E. coli BL21 (DE3) (Novagen) was transformed with theexpression vector (pAS45 and its derivatives). Cells were grown in M9minimal media and M9 media supplemented with Bactotryptone (Difco)containing ampicillin (200 μg/ml). For isotopic labeling, ¹⁵N NH₄Clreplaced unlabeled NH₄Cl in the media. 500 ml medium in a 2 liter baffleflask was inoculated with 10 ml of overnight culture and agitated at 37°C. IPTG was added at a final concentration of 1 mM to initiate proteinexpression when OD (600 nm) reaches one. The cells were harvested bycentrifugation 3 hours after the addition of IPTG and kept frozen at−70° C. until used.

Proteins were purified as follows. Cells were suspended in 5 ml/(g cell)of Tris (50 mM, pH 7.6) containing phenylmethylsulfonyl fluoride (1 mM).Hen egg lysozyme (Sigma) was added to a final concentration of 0.5mg/ml. After incubating the solution for 30 minutes at 37° C., it wassonicated three times for 30 seconds on ice. Cell debris was removed bycentrifugation. Concentrated sodium chloride was added to the solutionto a final concentration of 0.5 M. The solution was applied to a Hi-Trapchelating column (Pharmacia) preloaded with nickel and equilibrated inthe Tris buffer containing sodium chloride (0.5 M). After washing thecolumn with the buffer, histag-Fn3 was eluted with the buffer containing500 mM imidazole. The protein was further purified using a ResourceScolumn (Pharmacia) with a NaCl gradient in a sodium acetate buffer (20mM, pH 4.6).

With the GKKGK (SEQ ID NO:109) tail, the solubility of the Ubi4 proteinwas increased to over 1 mM at low pH and up to ˜50 μM at neutral pH.Therefore, further analyses were performed on Ubi4 with this C-terminalextension (hereafter referred to as Ubi4-K). It has been reported thatthe solubility of a minibody could be significantly improved by additionof three Lys residues at the N- or C-termini (Bianchi et al., 1994). Inthe case of protein Rop, a non-structured C-terminal tail is critical inmaintaining its solubility (Smith et al., 1995).

Oligomerization states of the Ubi4 protein were determined using a sizeexclusion column. The wild-type Fn3 protein was monomeric at low andneutral pH's. However, the peak of the Ubi4-K protein was significantlybroader than that of wild-type Fn3, and eluted after the wild-typeprotein. This suggests interactions between Ubi4-K and the columnmaterial, precluding the use of size exclusion chromatography todetermine the oligomerization state of Ubi4. NMR studies suggest thatthe protein is monomeric at low pH.

The Ubi4-K protein retained a binding affinity to ubiquitin as judged byELISA (FIG. 15 d). However, an attempt to determine the dissociationconstant using a biosensor (Affinity Sensors, Cambridge, U.K.) failedbecause of high background binding of Ubi4-K-Fn3 to the sensor matrix.This matrix mainly consists of dextran, consistent with the observationthat interactions between Ubi4-K interacts with the cross-linked dextranof the size exclusion column.

Example XVII Stability Measurements of Monobodies

Guanidine hydrochloride (GuHCl)-induced unfolding and refoldingreactions were followed by measuring tryptophan fluorescence.Experiments were performed on a Spectronic AB-2 spectrofluorometerequipped with a motor-driven syringe (Hamilton Co.). The cuvettetemperature was kept at 30° C. The spectrofluorometer and the syringewere controlled by a single computer using a home-built interface. Thissystem automatically records a series of spectra following GuHCltitration. An experiment started with a 1.5 ml buffer solutioncontaining 5 μM protein. An emission spectrum (300-400 nm; excitation at290 nm) was recorded following a delay (3-5 minutes) after eachinjection (50 or 100 μl) of a buffer solution containing GuHCl. Thesesteps were repeated until the solution volume reached the full capacityof a cuvette (3.0 ml). Fluorescence intensities were normalized asratios to the intensity at an isofluorescent point which was determinedin separate experiments. Unfolding curves were fitted with a two-statemodel using a nonlinear least-squares routine (Santoro & Bolen, 1988).No significant differences were observed between experiments with delaytimes (between an injection and the start of spectrum acquisition) of 2minutes and 10 minutes, indicating that the unfolding/refoldingreactions reached close to an equilibrium at each concentration pointwithin the delay times used.

Conformational stability of Ubi4-K was measured using above-describedGuHCl-induced unfolding method. The measurements were performed undertwo sets of conditions; first at pH 3.3 in the presence of 300 mM sodiumchloride, where Ubi4-K is highly soluble, and second in TBS, which wasused for library screening. Under both conditions, the unfoldingreaction was reversible, and we detected no signs of aggregation orirreversible unfolding. FIG. 16 shows unfolding transitions of Ubi4-Kand wild-type Fn3 with the N-terminal (his)₆ tag and the C-terminalsolubility tag. The stability of wild-type Fn3 was not significantlyaffected by the addition of these tags. Parameters characterizing theunfolding transitions are listed in Table 8.

TABLE 8 Stability parameters for Ubi4 and wild-type Fn3 as determined byGuHCl-induced unfolding Protein ΔG₀ (kcal mol⁻¹) m_(G) (kcal mol⁻¹ M⁻¹)Ubi4 (pH 7.5) 4.8 ± 0.1 2.12 ± 0.04 Ubi4 (pH 3.3) 6.5 ± 0.1 2.07 ± 0.02Wild-type (pH 7.5) 7.2 ± 0.2 1.60 ± 0.04 Wild-type (pH 3.3) 11.2 ± 0.1 2.03 ± 0.02 ΔG₀ is the free energy of unfolding in the absence ofdenaturant; m_(G) is the dependence of the free energy of unfolding onGuHCl concentration. For solution conditions, see FIG. 4 caption.Though the introduced mutations in the two loops certainly decreased thestability of Ubi4-K relative to wild-type Fn3, the stability of Ubi4remains comparable to that of a “typical” globular protein. It shouldalso be noted that the stabilities of the wild-type and Ubi4-K proteinswere higher at pH 3.3 than at pH 7.5.

The Ubi4 protein had a significantly reduced solubility as compared tothat of wild-type Fn3, but the solubility was improved by the additionof a solubility tail. Since the two mutated loops include the onlydifferences between the wild-type and Ubi4 proteins, these loops must bethe origin of the reduced solubility. At this point, it is not clearwhether the aggregation of Ubi4-K is caused by interactions between theloops, or by interactions between the loops and the invariable regionsof the Fn3 scaffold.

The Ubi4-K protein retained the global fold of Fn3, showing that thisscaffold can accommodate a large number of mutations in the two loopstested. Though the stability of the Ubi4-K protein is significantlylower than that of the wild-type Fn3 protein, the Ubi4 protein still hasa conformational stability comparable to those for small globularproteins. The use of a highly stable domain as a scaffold is clearlyadvantageous for introducing mutations without affecting the global foldof the scaffold. In addition, the GuHCl-induced unfolding of the Ubi4protein is almost completely reversible. This allows the preparation ofa correctly folded protein even when a Fn3 mutant is expressed in amisfolded form, as in inclusion bodies. The modest stability of Ubi4 inthe conditions used for library screening indicates that Fn3 variantsare folded on the phage surface. This suggests that a Fn3 clone isselected by its binding affinity in the folded form, not in a denaturedform. Dickinson et al., proposed that Val 29 and Arg 30 in the BC loopstabilize Fn3. Val 29 makes contact with the hydrophobic core, and Arg30 forms hydrogen bonds with Gly 52 and Val 75. In Ubi4-Fn3, Val 29 isreplaced with Arg, while Arg 30 is conserved. The FG loop was alsomutated in the library. This loop is flexible in the wild-typestructure, and shows a large variation in length among human Fn3 domains(Main et al., 1992). These observations suggest that mutations in the FGloop may have less impact on stability. In addition, the N-terminal tailof Fn3 is adjacent to the molecular surface formed by the BC and FGloops (FIGS. 1 and 17) and does not form a well-defined structure.Mutations in the N-terminal tail would not be expected to have strongdetrimental effects on stability. Thus, residues in the N-terminal tailmay be good sites for introducing additional mutations.

Example XVIII NMR Spectroscopy of Ubi4-Fn3

Ubi4-Fn3 was dissolved in [²H]-Gly HCl buffer (20 mM, pH 3.3) containingNaCl (300 mM) using an Amicon ultrafiltration unit. The final proteinconcentration was 1 mM. NMR experiments were performed on a Varian UnityINOVA 600 spectrometer equipped with a triple-resonance probe withpulsed field gradient. The probe temperature was set at 30° C. HSQC,TOCSY-HSQC and NOESY-HSQC spectra were recorded using publishedprocedures (Kay et al., 1992; Zhang et al., 1994). NMR spectra wereprocessed and analyzed using the NMRPipe and NMRView software (Johnson &Blevins, 1994; Delaglio et al., 1995) on UNIX workstations.Sequence-specific resonance assignments were made using standardprocedures (Wüthrich, 1986; Clore & Gronenborn, 1991). The assignmentsfor wild-type Fn3 (Baron et al., 1992) were confirmed using a¹⁵N-labeled protein dissolved in sodium acetate buffer (50 mM, pH 4.6)at 30° C.

The three-dimensional structure of Ubi4-K was characterized using thisheteronuclear NMR spectroscopy method. A high quality spectrum could becollected on a 1 mM solution of ¹⁵N-labeled Ubi4 (FIG. 17 a) at low pH.The linewidth of amide peaks of Ubi4-K was similar to that of wild-typeFn3, suggesting that Ubi4-K is monomeric under the conditions used.Complete assignments for backbone ¹H and ¹⁵N nuclei were achieved usingstandard ¹H, ¹⁵N double resonance techniques, except for a row of Hisresidues in the N-terminal (His)₆ tag. There were a few weak peaks inthe HSQC spectrum which appeared to originate from a minor speciescontaining the N-terminal Met residue. Mass spectroscopy analysis showedthat a majority of Ubi4-K does not contain the N-terminal Met residue.FIG. 17 shows differences in ¹HN and ¹⁵N chemical shifts between Ubi4-Kand wild-type Fn3. Only small differences are observed in the chemicalshifts, except for those in and near the mutated BC and FG loops. Theseresults clearly indicate that Ubi4-K retains the global fold of Fn3,despite the extensive mutations in the two loops. A few residues in theN-terminal region, which is close to the two mutated loops, also exhibitsignificant chemical differences between the two proteins. An HSQCspectrum was also recorded on a 50 μM sample of Ubi4-K in TBS. Thespectrum was similar to that collected at low pH, indicating that theglobal conformation of Ubi4 is maintained between pH 7.5 and 3.3.

Example XIX Stabilization of Fn3 Domain by Removing UnfavorableElectrostatic Interactions on the Protein Surface Introduction

Increasing the conformational stability of a protein by mutation is amajor interest in protein design and biotechnology. Thethree-dimensional structures of proteins are stabilized by combinationof different types of forces. The hydrophobic effect, van der Waalsinteractions and hydrogen bonds are known to contribute to stabilize thefolded state of proteins (Kauzmann, W. (1959) Adv. Prot. Chem. 14, 1-63;Dill, K. A. (1990) Biochemistry 29, 7133-7155; Pace, C. N., Shirley, B.A., McNutt, M. & Gajiwala, K. (1996) Faseb J 10, 75-83). Thesestabilizing forces primarily originate from residues that are wellpacked in a protein, such as those that constitute the hydrophobic core.Because a change in the protein core would induce a rearrangement ofadjacent moieties, it is difficult to improve protein stability byincreasing these forces without massive computation (Malakauskas, S. M.& Mayo, S. L. (1998) Nat Struct Biol 5, 470-475). Ion pairs betweencharged groups are commonly found on the protein surface (Creighton, T.E. (1993) Proteins: structures and molecular properties, Freeman, NewYork), and an ion pair could be introduced to a protein with smallstructural perturbations. However, a number of studies have demonstratedthat the introduction of an attractive electrostatic interaction, suchas an ion pair, on protein surface has small effects on stability(Dao-pin, S., Sauer, U., Nicholson, H. & Matthews, B. W. (1991)Biochemistry 30, 7142-7153; Sali, D., Bycroft, M. & Fersht, A. R. (1991)J. Mol. Biol. 220, 779-788). A large desolvation penalty and the loss ofconformational entropy of amino acid side chains oppose the favorableelectrostatic contribution (Yang, A.-S. & Honig, B. (1992) Curr. Opin.Struct. Biol. 2, 40-45; Hendsch, Z. S. & Tidor, B. (1994) Protein Sci.3, 211-226). Recent studies demonstrated that repulsive electrostaticinteractions on the protein surface, in contrast, may significantlydestabilize a protein, and that it is possible to improve proteinstability by optimizing surface electrostatic interactions (Loladze, V.V., Ibarra-Molero, B., Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999)Biochemistry 38, 16419-16423; Perl, D., Mueller, U., Heinemann, U. &Schmid, F. X. (2000) Nat Struct Biol 7, 380-383; Spector, S., Wang, M.,Carp, S. A., Robblee, J., Hendsch, Z. S., Fairman, R., Tidor, B. &Raleigh, D. P. (2000) Biochemistry 39, 872-879; Grimsley, G. R., Shaw,K. L., Fee, L. R., Alston, R. W., Huyghues-Despointes, B. M., Thurlkill,R. L., Scholtz, J. M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849). Inthe present experiments, the inventor improved protein stability bymodifying surface electrostatic interactions.

During the characterization of monobodies it was found that theseproteins, as well as wild-type FNfn10, are significantly more stable atlow pH than at neutral pH (Koide, A., Bailey, C. W., Huang, X. & Koide,S. (1998) J. Mol. Biol. 284, 1141-1151). These observations indicatethat changes in the ionization state of some moieties in FNfn10 modulatethe conformational stability of the protein, and suggest that it mightbe possible to enhance the conformational stability of FNfn10 at neutralpH by adjusting electrostatic properties of the protein. Improving theconformational stability of FNfn10 will also have practical importancein the use of FNfn10 as a scaffold in biotechnology applications.

Described below are experiments that detailed characterization of the pHdependence of FNfn10 stability, identified unfavorable interactionsbetween side chain carboxyl groups, and improved the conformationalstability of FNfn10 by point mutations on the surface. The resultsdemonstrate that the surface electrostatic interactions contributesignificantly to protein stability, and that it is possible to enhanceprotein stability by rationally modulating these interactions.

Experimental Procedures Protein Expression and Purification

The wild-type protein used for the NMR studies contained residues 1-94of FNfn10 (residue numbering is according to FIG. 2(a) of Koide et al.(Koide, A., Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol.284, 1141-1151)), and additional two residues (Met-Gln) at theN-terminus (these two residues are numbered −2 and −1, respectively).The gene coding for the protein was inserted in pET3a (Novagen, WI).Eschericha coli BL21 (DE3) transformed with the expression vector wasgrown in the M9 minimal media supplemented with ¹³C-glucose and¹⁵N-ammonium chloride (Cambridge Isotopes) as the sole carbon andnitrogen sources, respectively. Protein expression was induced asdescribed previously (Koide, A., Bailey, C. W., Huang, X. & Koide, S.(1998) J. Mol. Biol. 284, 1141-1151). After harvesting the cells bycentrifuge, the cells were lysed as described (Koide, A., Bailey, C. W.,Huang, X. & Koide, S. (1998) J. Mol. Biol. 284, 1141-1151). Aftercentrifugation, supernatant was dialyzed against 10 mM sodium acetatebuffer (pH 5.0), and the protein solution was applied to a SP-SepharoseFastFlow column (Amersham Pharmacia Biotech), and FN3 was eluted with agradient of sodium chloride. The protein was concentrated using anAmicon concentrator using YM-3 membrane (Millipore).

The wild-type protein used for the stability measurements contained anN-terminal histag (MGSSHHHHHHSSGLVPRGSH) (SEQ ID NO:114) and residues−2-94 of FNfn10. The gene for FN3 described above was inserted in pET15b(Novagen). The protein was expressed and purified as described (Koide,A., Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol. 284,1141-1151). The wild-type protein used for measurements of the pHdependence shown in FIG. 22 contained Arg 6 to Thr mutation, which hadoriginally been introduced to remove a secondary thrombin cleavage site(Koide, A., Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol.284, 1141-1151). Because Asp 7, which is adjacent to Arg 6, was found tobe critical in the pH dependence of FN3 stability as detailed underResults, subsequent studies were performed using the wild-type, Arg 6,background. The genes for the D7N and D7K mutants were constructed usingstandard polymerase chain reactions, and inserted in pET15b. Theseproteins were prepared in the same manner as for the wild-type protein.¹³C, ¹⁵N-labeled proteins for pK_(a) measurements were prepared asdescribed above, and the histag moiety was not removed from theseproteins.

Chemical Denaturation Measurements

Proteins were dissolved to a final concentration of 5 μM in 10 mM sodiumcitrate buffer at various pH containing 100 mM sodium chloride.Guanidine HCl (GuHCl)-induce unfolding experiments were performed asdescribed previously (Koide, A., Bailey, C. W., Huang, X. & Koide, S.(1998) J. Mol. Biol. 284, 1141-1151; Koide, S., Bu, Z., Risal, D., Pham,T.-N., Nakagawa, T., Tamura, A. & Engelman, D. M. (1999) Biochemistry38, 4757-4767). GuHCl concentration was determined using an Abberefractometer (Spectronic Instruments) as described (Pace, C. N. &Sholtz, J. M. (1997) in Protein structure. A practical approach(Creighton, T. E., Ed.) Vol. pp 299-321, IRL Press, Oxford). Data wereanalyzed according to the two-state model as described (Koide, A.,Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol. 284,1141-1151; Santoro, M. M. & Bolen, D. W. (1988) Biochemistry 27,8063-8068.).

Thermal Denaturation Measurements

Proteins were dissolved to a final concentration of 5 μM in 20 mM sodiumphosphate buffer (pH 7.0) containing 0.1 or 1 M sodium chloride or in 20mM glycine HCl buffer (pH 2.4) containing 0.1 or 1 M sodium chloride.Additionally 6.3 M urea was included in all solutions to ensurereversibility of the thermal denaturation reaction. In the absence ofurea it was found that denatured FNfn10 adheres to quartz surface, andthat the thermal denaturation reaction was irreversible. Circulardichroism measurements were performed using a Model 202 spectrometerequipped with a Peltier temperature controller (Aviv Instruments). Acuvette with a 0.5-cm pathlength was used. The ellipticity at 227 nm wasrecorded as the sample temperature was raised at a rate of approximately1° C. per minute. Because of decomposition of urea at high temperature,the pH of protein solutions tended to shift upward during an experiment.The pH of protein solution was measured before and after each thermaldenaturation measurement to ensure that a shift no more than 0.2 pH unitoccurred in each measurement. At pH 2.4, two sections of a thermaldenaturation curve (30-65° C. and 60-95° C.) were acquired from separatesamples, in order to avoid a large pH shift. The thermal denaturationdata were fit with the standard two-state model (Pace, C. N. & Sholtz,J. M. (1997) in Protein structure. A practical approach (Creighton, T.E., Ed.) Vol. pp 299-321, IRL Press, Oxford):

ΔG(T)=ΔH _(m)(1−T/T _(m))−ΔC _(P)[(T _(m) −T)+T ln(T/T _(m))]

where ΔG(T) is the Gibbs free energy of unfolding at temperature T,ΔH_(m) is the enthalpy change upon unfolding at the midpoint of thetransition, T_(m), and ΔC_(P) is the heat capacity change uponunfolding. The value for ΔC_(p) was fixed at 1.74 kcal mol⁻¹ K⁻¹,according to the approximation of Myers et al. (Myers, J. K., Pace, C.N. & Scholtz, J. M. (1995) Protein Sci. 4, 2138-2148). Most of thedatasets taken in the presence of 1 M NaCl did not have a sufficientbaseline for the unfolded state, and thus it was assumed the slope ofthe unfolded baseline in the presence of 1 M NaCl to be identical tothat determined in the presence of 0.1 M NaCl.

NMR Spectroscopy

NMR experiments were performed at 30° C. on an INOVA 600 spectrometer(Varian Instruments). The C(CO)NH experiment (Grzesiek, S., Anglister,J. & Bax, A. (1993) J. Magn. Reson. B 101, 114-119) and the CBCACOHAexperiment (Kay, L. E. (1993) J. Am. Chem. Soc. 115, 2055-2057) werecollected on a [¹³C, ¹⁵N]-wild-type FNfn10 sample (1 mM) dissolved in 50mM sodium acetate buffer (pH 4.6) containing 5% (v/v) deuterium oxide,using a Varian 5 mm triple resonance probe with pulsed field gradient.The carboxyl ¹³C resonances were assigned based on the backbone ¹H, ¹³Cand ¹⁵N resonance assignments of FNfn10 (Baron, M., Main, A. L.,Driscoll, P. C., Mardon, H. J., Boyd, J. & Campbell, I. D. (1992)Biochemistry 31, 2068-2073). pH titration of carboxyl resonances wereperformed on a 0.3 mM FNfn10 sample dissolved in 10 mM sodium citratecontaining 100 mM sodium chloride and 5% (v/v) deuterium oxide. An 8 mmtriple-resonance, pulse-field gradient probe (Nanolac Corporation) wasused for pH titration. Two-dimensional H(C)CO spectra were collectedusing the CBCACOHA pulse sequence as described previously (McIntosh, L.P., Hand, G., Johnson, P. E., Joshi, M. D., Koerner, M., Plesniak, L.A., Ziser, L., Wakarchuk, W. W. & Withers, S. G. (1996) Biochemistry 35,9958-9966). Sample pH was changed by adding small aliquots ofhydrochloric acid, and pH was measured before and after taking NMR data.¹H, ¹⁵N-HSQC spectra were taken as described previously (Kay, L. E.,Keifer, P. & Saarinen, T. (1992) J. Am. Chem. Soc. 114, 10663-10665).NMR data were processed using the NMRPipe package (Delaglio, F.,Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. & Bax, A. (1995) J.Biomol. NMR 6, 277-293), and analyzed using the NMRView software(Johnson, B. A. & Blevins, R. A. (1994) J. Biomol. NMR 4, 603-614).

NMR titration curves of the carboxyl ¹³C resonances were fit to theHenderson-Hasselbalch equation to determine pK_(a)'s:

δ(pH)=(δ_(acid)+δ_(base)10^((pH-pK) ^(a) ⁾)/(1+10^((pH-pKa)))

where δ is the measured chemical shift, δ_(acid) is the chemical shiftassociated with the protonated state, δ_(base) is the chemical shiftassociated with the deprotonated state, and pK_(a) is the pK_(a) valuefor the residue. Data were also fit to an equation with two ionizablegroups:

δ(pH)=(δ_(AH2′)+δ_(AH)10^((pH-pK) ^(a1) ⁾+δ_(A)10^((2pH-pK) ^(a1) ^(pK)^(a2) ⁾)/(1+10^((pH-pK) ^(a1) ⁾+10^((2pH-pK) ^(a1) ^(-pK) ^(a) ⁾)

where δ_(AH2), δ_(AH) and δ_(A) are the chemical shifts associated withthe fully protonated, singularly protonated and deprotonated states,respectively, and pK_(a1) and pK_(a2) are pK_(a)'s associated with thetwo ionization steps. Data fitting was performed using the nonlinearleast-square regression method in the program Igor Pro (WaveMetrix, OR)on a Macintosh computer.

Results pH Dependence of FNfn10 Stability

Previously, it was found that FNfn10 is more stable at acidic pH than atneutral pH (Koide, A., Bailey, C. W., Huang, X. & Koide, S. (1998) J.Mol. Biol. 284, 1141-1151). In the present experiments, the pHdependence of its stability was further characterized. Because of itshigh stability, FNfn10 could not be fully denatured in urea at 30° C.Thus GuHCl-induced chemical denaturation (FIG. 18) was used. Thedenaturation reaction was fully reversible under all conditions tested.In order to minimize errors caused by extrapolation, the free energy ofunfolding at 4 M GuHCl was used for comparison (FIG. 18). The stabilityincreased as the pH was lowered, with apparent plateaus at both ends ofthe pH range. The pH dependence curve has an apparent transitionmidpoint near pH 4. In addition, a gradual increase in the m value, thedependence of the unfolding free energy on denaturant concentration wasnoted. Pace et al. reported a similar pH dependence of the m value forbarnase (Pace, C. N., Laurents, D. V. & Erickson, R. E. (1992)Biochemistry 31, 2728-2734). These results indicate that FNfn10 containsinteractions that stabilize the protein at low pH, or those thatdestabilize it at neutral pH. The results also suggest that byidentifying and altering the interactions that give rise to the pHdependence, one may be able to improve the stability of FNfn10 atneutral pH to a degree similar to that found at low pH.

Determination of pK_(a)'s of the Side Chain Carboxyl Groups in Wild-TypeFNfn10

The pH dependence of FNfn10 stability suggests that amino acids withpK_(a) near 4 are involved in the observed transition. The carboxylgroups of Asp and Glu generally have pK_(a) in this range (Creighton, T.E. (1993) Proteins: structures and molecular properties, Freeman, NewYork). It is well known that if a carboxyl group has unfavorable (i.e.destabilizing) interactions in the folded state, its pK_(a) is shiftedto a higher value from its unperturbed value (Yang, A.-S. & Honig, B.(1992) Curr. Opin. Struct. Biol. 2, 40-45). If a carboxyl group hasfavorable interactions in the folded state, it has a lower pK_(a). Thus,the pK_(a) values of all carboxylates in FNfn10 using heteronuclear NMRspectroscopy were determined in order to identify stabilizing anddestabilizing interactions involving carboxyl groups.

First, the ¹³C resonance for the carboxyl carbon of each Asp and Gluresidue in FN3 was assigned (FIG. 19). Next, pH titration of the ¹³Cresonances for these groups was performed (FIG. 20). Titration curvesfor Asp 3, 67 and 80, and Glu 38 and 47 could be fit well with theHenderson-Hasselbalch equation with a single pK_(a). The pK_(a) valuesfor these residues (Table 9) are either close to or slightly lower thantheir respective unperturbed values (3.8-4.1 for Asp, and 4.1-4.6 forGlu (Kuhlman, B., Luisi, D. L., Young, P. & Raleigh, D. P. (1999)Biochemistry 38, 4896-4903)), indicating that these carboxyl groups areinvolved in neutral or slightly favorable electrostatic interactions inthe folded state.

TABLE 9 pK_(a) values for Asp and Glu residues in FN3¹. Protein ResidueWild-Type D7N D7K E9 3.84, 5.40² 4.98 4.53 E38 3.79 3.87 3.86 E47 3.943.99 3.99 D3 3.66 3.72 3.74 D7 3.54, 5.54² — — D23 3.54, 5.25² 3.68 3.82D67 4.18 4.17 4.14 D80 3.40 3.49 3.48 ¹The standard deviations in thepK_(a) values are less than 0.05 pH units for those fit with a singlepK_(a) and less than 0.15 pH unit for those with two pK_(a)'s. ²Data forE9, D7 and D23 were fit with a transition curve with two pK_(a) values.

The titration curves for Asp 7 and 23, and Glu 9 were fit better withthe Henderson-Hasselbalch equation with two pK_(a) values, and one ofthe two pK_(a) values for each were shifted higher than the respectiveunperturbed values (FIG. 19B). The titration curves with two apparentpK_(a) values of these carboxyl groups may be due to influence of anionizable group in the vicinity. In the three-dimensional structure ofFNfn10 (Main, A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I.D. (1992) Cell 71, 671-678), Asp 7 and 23, and Glu 9 form a patch on thesurface (FIG. 21), with Asp 7 centrally located in the patch. Thus, itis reasonable to expect that these residues influence each other'sionization profile. In order to identify which of the three residueshave a highly upshifted pK_(a), the H(C)CO spectrum of the protein in99% D₂O buffer at pH* 5.0 (direct pH meter reading) was then collected.Asp 23 and Glu 9 showed larger deuterium isotope shifts (0.33 and 0.32ppm, respectively) than Asp 7 (0.18 ppm). These results show that Asp 23and Glu 9 are protonated to a greater degree than Asp 7. Thus, weconcluded that Asp 23 and Glu 9 have highly upshifted pK_(a)'s, due tostrong influence of Asp 7.

Mutational Analysis

The spatial proximity of Asp 7 and 23, and Glu 9 explains theunfavorable electrostatic interactions in FNfn10 identified in thisstudy. At low pH where these residues are protonated and neutral, therepulsive interactions are expected to be mostly relieved. Thus, itshould be possible to improve the stability of FNfn10 at neutral pH, byremoving the electrostatic repulsion between these three residues.Because Asp 7 is centrally located among the three residues, it wasdecided to mutate Asp 7. Two mutants, D7N and D7K were prepared. Theformer neutralizes the negative charge with a residue of virtuallyidentical size. The latter places a positive charge at residue 7 andincreases the size of the side chain.

The ¹H, ¹⁵N-HSQC spectra of the two mutant proteins were nearlyidentical to that of the wild-type protein, indicating that thesemutations did not cause large structural perturbations (data not shown).The degrees of stability of the mutant proteins were then characterizedusing thermal and chemical denaturation measurements. Thermaldenaturation measurements were performed initially with 100 mM sodiumchloride, and 6.3 M urea was included to ensure reversible denaturationand to decrease the temperature of the thermal transition. All theproteins were predominantly folded in 6.3 M urea at room temperature.All the proteins underwent a cooperative transition, and the two mutantswere found to be significantly more stable than the wild type at neutralpH (FIG. 22 and Table 10). Furthermore, these mutations almosteliminated the pH dependence of the conformational stability of FNfn10.These results confirmed that destabilizing interactions involving Asp 7in wild-type FNfn10 at neutral pH are the primary cause of the pHdependence.

TABLE 10 The midpoint of thermal denaturation (in ° C.) of wild-type andmutant FN3 in the presence of 6.3M urea. pH 2.4 pH 7.0 Protein 0.1M NaCl1M NaCl 0.1M NaCl 1M NaCl wild type 72 82 62 70 D7N 68 82 69 80 D7K 6977 70 78 The error in the midpoints for the 0.1M NaCl data is ±0.5° C.Because most of the 1M NaCl data did not have a sufficient baseline forthe denatured state, the error in the midpoints for these data wasestimated to be ±2° C.

The effect of increased sodium chloride concentration on theconformational stability of the wild type and the two mutant proteinswas next investigated. All proteins were more stable in 1 M sodiumchloride than in 0.1 M sodium chloride (FIG. 22). The increase of thesodium chloride concentration elevated the T_(m) of the mutant proteinsby approximately 10° C. at both acidic and neutral pH (Table 10).Remarkably the wild-type protein was also equally stabilized at both pH,although it contains unfavorable interactions among the carboxyl groupsat neutral pH but not at acidic pH.

Chemical denaturation of FNfn10 proteins was monitored usingfluorescence emission from the single Trp residue of FNfn10 (FIG. 23).The free energies of unfolding at pH 6.0 and 4 M GuHCl were determinedto be 1.1 (±0.3), 1.7 (±0.2) and 1.4 (±0.1) kcal/mol for the wild type,D7N and D7K, respectively, indicating that the two mutations alsoincreased the conformational stability against chemical denaturation.

Determination of the pK_(a)'s of the Side Chain Carboxyl Groups in theMutant Proteins

The ionization properties of carboxyl groups in the two mutant proteinswas investigated. The 2D H(C)CO spectra of the mutant proteins at thehigh and low ends of the pH titration (pH ˜7 and ˜1.5, respectively)were nearly identical to the respective spectra of the wild type, exceptfor the loss of the cross peaks for Asp 7 (data not shown). Thissimilarity allowed for an unambiguous assignment of resonances of themutants, based on the assignments for wild-type FNfn10. The pH titrationexperiments revealed that, except for Glu 9 and Asp 23, the behaviors ofAsp and Glu carboxyl groups are very close to their counterparts in thewild-type protein (FIG. 24 Panels A, C, D, F and G, and Table 9),indicating that the two mutations have marginal effects on theelectrostatic environments for these carboxylates. In contrast, thetitration curves for E9 and D23 show significant changes upon mutation(FIG. 24 Panels B and E). The pK_(a) of D23 was lowered by more than 1.6and 1.4 pH units in the D7N and D7K mutants, respectively. These resultsclearly show that the repulsive interaction between D7 and D23contributes to the increase in pK_(a) of Asp 23 in the wild-typeprotein, and that it was eliminated by the neutralization of thenegative charge at residue 7. The pK_(a) of Glu 9 was reduced by 0.4 pHunit by the D7N mutation, while it was decreased by 0.8 pH units in theD7K mutant. The greater reduction of Glu 9 pK_(a) by the D7K mutationsuggests that there is a favorable interaction between Lys 7 and Glu 9in this mutant protein.

Discussion

The present inventor has identified unfavorable electrostaticinteractions in FNfn10, and improved its conformational stability bymutations on the protein surface. The results demonstrate that repulsiveinteractions between like charges on protein surface significantlydestabilize a protein. The results are also consistent with recentreports by other groups (Loladze, V. V., Ibarra-Molero, B.,Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999) Biochemistry 38,16419-16423; Perl, D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000)Nat Struct Biol 7, 380-383; Spector, S., Wang, M., Carp, S. A., Robblee,J., Hendsch, Z. S., Fairman, R., Tidor, B. & Raleigh, D. P. (2000)Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L. R.,Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L., Scholtz, J.M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849), in which proteinstability was improved by eliminating unfavorable electrostaticinteractions on the surface. In these studies, candidates for mutationswere identified by electrostatic calculations (Loladze, V. V.,Ibarra-Molero, B., Sanchez-Ruiz, J M. & Makhatadze, G. I. (1999)Biochemistry 38, 16419-16423; Spector, S., Wang, M., Carp, S. A.,Robblee, J., Hendsch, Z. S., Fairman, R., Tidor, B. & Raleigh, D. P.(2000) Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L.R., Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L.,Scholtz, J. M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849) or bysequence comparison of homologous proteins with different stability(Peri, D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000) Nat StructBiol 7, 380-383). The present strategy using pK_(a) determination usingNMR has both advantages and disadvantages over the other strategies. Thepresent method directly identifies residues that destabilize a protein.Also it does not depend on the availability of the high-resolutionstructure of the protein of interest. Electrostatic calculations mayhave large errors due to the flexibility of amino acid side chains onthe surface, and the uncertainty in the dielectric constant on theprotein surface and in the protein interior. For example, in the NMRstructure of FNfn10 (Main, A. L., Harvey, T. S., Baron, M., Boyd, J. &Campbell, I. D. (1992) Cell 71, 671-678), the root mean squareddeviations among 16 model structures for the O^(∈) atom of Glu residuesare 1.2-2.4 Å, and those for Lys N^(ζ) atoms are 1.5-3.1 Å. Suchuncertainties in atom position can potentially cause large differencesin calculation results. On the other hand, the present strategy requiresthe NMR assignments for carboxyl residues, and NMR measurements over awide pH range. Although recent advances in NMR spectroscopy have made itstraightforward to obtain resonance assignments for a small protein,some proteins may not be sufficiently soluble over the desired pH range.In addition, knowledge of the pK_(a) values of ionizable groups in thedenatured state is necessary for accurately evaluating contributions ofindividual residues to stability (Yang, A.-S. & Honig, B. (1992) Curr.Opin. Struct. Biol. 2, 40-45). Kuhlman et al. (Kuhlman, B., Luisi. D.L., Young, P. & Raleigh, D. P. (1999) Biochemistry 38, 4896-4903) showedthat pK_(a)'s of carboxylates in the denatured state has a considerablylarge range than those obtained from small model compounds. Despitethese limitations, the present method is applicable to many proteins.

The inventor showed that the unfavorable interactions involving thecarboxyl groups of Asp 7, Glu 9 and Asp23 were no longer present ifthese groups are protonated at low pH or if Asp 7 was replaced with Asnor Lys. The similarity in the measured stability of the mutants and thewild type at low pH (Table 10) suggests that no other factorssignificantly contribute to the pH dependence of FNfn10 stability andthat the mutations caused minimal structural perturbations. The littlestructural perturbation was expected, since the carboxyl groups of thesethree residues are at least 50% exposed to the solvent, based on thesolvent accessible surface area calculation on the NMR structure (Main,A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I. D. (1992) Cell71, 671-678).

The difference in thermal stability of the wild-type protein betweenacidic and neutral pH persisted in 1 M sodium chloride (Table 10).Likewise, the wild-type protein exhibited a large pH-dependence instability in 4 M GuHCl (FIG. 18). Furthermore, upon the increase in thesodium chloride concentration from 0.1 to 1.0 M, the T_(m) of thewild-type and mutant proteins all increased by ˜10° C., which is in thesame magnitude as the change in T_(m) of the wild type by the pH shift.These data indicate that the unfavorable interactions identified in thisstudy were not effectively shielded in 1 M NaCl or in 4 M GuHCl. Becausethe effect of increased sodium chloride was uniform, this stabilizationeffect of sodium chloride is likely due to the nonspecific salting-outeffect (Timasheff, S. N. (1992) Curr. Op. Struct. Biol. 2, 35-39). Othergroups also reported little shielding effect of salts on electrostaticinteractions (Perutz, M. F., Gronenborn, A. M., Clore, G. M., Fogg, J.H. & Shih, D. T. (1985) J Mol Biol 183, 491-498; Hendsch, Z. S.,Jonsson, T., Sauer, R. T. & Tidor, B. (1996) Biochemistry 35,7621-7625). Electrostatic interactions are often thought to diminishwith increasing ionic strength, particularly if the site of interactionis highly exposed. Accordingly, the present data at neutral pH (Table10) showing no difference in the salt sensitivity between the wild typeand the mutants could be interpreted as Asp 7 not being responsible fordestabilizing electrostatic interactions. Although the reason for thissalt insensitivity is not yet clear, the present results provide acautionary note on concluding the presence and absence of electrostaticinteractions solely based on salt concentration dependence.

The carboxyl triad (Asp 7 and 23, and Glu 9) is highly conserved inFNfn10 from nine different organisms that were available in the proteinsequence databank at National Center for Biotechnology Information(www.ncbi.nlm.nih.gov). In these FNfn10 sequences, Asp 9 is conservedexcept one case where it is replaced with Asn, and Glu 9 is completelyconserved. The position 23 is either Asp or Glu, preserving the negativecharge. As was discovered in this study, the interactions among theseresidues are destabilizing. Thus, their high conservation, despite theirnegative effects on stability, suggests that these residues havefunctional importance in the biology of fibronectin. In the structure ofa four-FN3 segment of human fibronectin (Leahy, D. J., Aukhil, I. &Erickson, H. P. (1996) Cell 84, 155-164), these residues are notdirectly involved in interactions with adjacent domains. Also theseresidues are located on the opposite face of FNfn10 from theintegrin-binding RGD sequence in the FG loop (FIG. 21). Therefore, it isnot clear why these destabilizing residues are almost completelyconserved in FNfn10. In contrast, no other FN3 domains in humanfibronectin contain this carboxyl triad (for a sequence alignment, seeref Main, A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I. D.(1992) Cell 71, 671-678). The carboxyl triad of FNfn10 may be involvedin important interactions that have not been identified to date.

Clarke et al. (Clarke, J., Hamill, S. J. & Johnson, C. M. (1997) J MolBiol 270, 771-778) reported that the stability of the third FN3 of humantenascin (TNfn3) increases as pH was decreased from 7 to 5. Althoughthey could not perform stability measurements below pH 5 due to proteinaggregation, the pH dependence of TNfn3 resembles that of FNfn10 shownin FIG. 18. TNfn3 does not contain the carboxylate triad at positions 7,9 and 23 (Leahy, D. J., Hendrickson, W. A., Aukhil, I. & Erickson, H. P.(1992) Science 258, 987-991), indicating that the destabilization ofTNfn3 at neutral pH is caused by a different mechanism from that forFNfn10. A visual inspection of the TNfn3 structure revealed that it hasa large number of carboxyl groups, and that Glu 834 and Asp 850(numbering according to ref Leahy, D. J., Hendrickson, W. A., Aukhil, I.& Erickson, H. P. (1992) Science 258, 987-991) forms a cross-strandpair. It will be interesting to examine whether altering this pair canincrease the stability of TNfn3.

In conclusion, a strategy has been described to experimentally identifyunfavorable electrostatic interactions on the protein surface andimprove the protein stability by relieving such interactions. Thepresent results have demonstrated that forming a repulsive interactionbetween carboxyl groups significantly destabilize a protein. This is incontrast to the small contributions of forming a solvent-exposed ionpair. Unfavorable electrostatic interactions on the surface seem quitecommon in natural proteins. Therefore, optimization of the surfaceelectrostatic properties provides a generally applicable strategy forincreasing protein stability (Loladze, V. V., Ibarra-Molero, B.,Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999) Biochemistry 38,16419-16423; Perl, D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000)Nat Struct Biol 7, 380-383; Spector, S., Wang, M., Carp, S. A., Robblee,J., Hendsch, Z. S., Fairman, R., Tidor, B. & Raleigh, D. P. (2000)Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L. R.,Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L., Scholtz, J.M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849). In addition,repulsive interactions between carboxylates can be exploited fordestabilizing undesirable, alternate conformations in protein design(“negative design”).

Example XX An Extension of the Carboxyl-Terminus of the MonobodyScaffold

The wild-type protein used for stability measurements is described underExample 19. The carboxyl-terminus of the monobody scaffold was extendedby four amino acid residues, namely, amino acid residues(Glu-Ile-Asp-Lys) (SEQ ID NO:119), which are the ones that immediatelyfollow FNfn10 of human fibronectin. The extension was introduced intothe FNfn10 gene using standard PCR methods. Stability measurements wereperformed as described under Example 19. The free energy of unfolding ofthe extended protein was 7.4 kcal mol⁻¹ at pH 6.0 and 30° C., very closeto that of the wild-type protein (7.7 kcal mol⁻¹). These resultsdemonstrate that the C-terminus of the monobody scaffold can be extendedwithout decreasing its stability.

The complete disclosure of all patents, patent documents andpublications cited herein are incorporated by reference as ifindividually incorporated. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

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What is claimed is:
 1. A fibronectin type III (Fn3) molecule, whereinthe Fn3 comprises a stabilizing mutation as compared to a wild-type Fn3.2. The Fn3 of claim 1, wherein the stabilizing mutation comprises atleast one aspartic acid (Asp) residue that has been deleted orsubstituted with at least one other amino acid residue.
 3. The Fn3 ofclaim 2, wherein Asp 7 or Asp 23, or both, have been deleted orsubstituted with at least one other amino acid residue.
 4. The Fn3 ofclaim 3, wherein Asp 7 or Asp 23, or both, have been substituted with anasparagine (Asn) or lysine (Lys) residue.
 5. The Fn3 of claim 1, whereinthe stabilizing mutation comprises at least one glutamic acid (Glu)residue that has been deleted or substituted with at least one otheramino acid residue.
 6. The Fn3 of claim 5, wherein Glu 9 has beendeleted or substituted with at least one other amino acid residue. 7.The Fn3 of claim 6, wherein Glu 9 has been substituted with anasparagine (Asn) or lysine (Lys) residue.
 8. The Fn3 of claim 2, whereinAsp 7, Asp 23, and Glu 9 have been deleted or substituted with at leastone other amino acid residue.
 9. A fibronectin type III (Fn3)polypeptide monobody comprising a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein one or more of the monobody loop region sequences vary bydeletion, insertion or replacement of at least two amino acids from thecorresponding loop region sequences in wild-type Fn3; wherein theβ-strand domains of the monobody have at least a 50% total amino acidsequence homology to the corresponding amino acid sequence of wild-typeFn3's β-strand domain sequences; and wherein the Fn3 comprises astabilizing mutation.
 10. An isolated nucleic acid molecule encoding theFn3 molecule of claim
 9. 11. An expression vector comprising anexpression cassette operably linked to the nucleic acid molecule ofclaim
 10. 12. A host cell comprising the vector of claim
 11. 13. Themonobody of claim 9, wherein at least one loop region is capable ofbinding to a specific binding partner (SBP) to form a polypeptide:SBPcomplex having a dissociation constant of less than 10⁻⁶ moles/liter.14. The monobody of claim 9, wherein at least one loop region is capableof catalyzing a chemical reaction with a catalyzed rate constant(k_(cat)) and an uncatalyzed rate constant (k_(uncat)) such that theratio of k_(cat)/k_(uncat) is greater than
 10. 15. The monobody of claim9, wherein one or more of the loop regions comprise amino acid residues:i) from 15 to 16 inclusive in an AB loop; ii) from 22 to 30 inclusive ina BC loop; iii) from 39 to 45 inclusive in a CD loop; iv) from 51 to 55inclusive in a DE loop; v) from 60 to 66 inclusive in an EF loop; andvi) from 76 to 87 inclusive in an FG loop.
 16. The monobody of claim 9,wherein the monobody loop region sequences vary from the wild-type Fn3loop region sequences by the deletion or replacement of at least 2 aminoacids.
 17. The monobody of claim 9, wherein the monobody loop regionsequences vary from the wild-type Fn3 loop region sequences by theinsertion of from 3 to 25 amino acids.
 18. An isolated nucleic acidmolecule encoding the polypeptide monobody of claim
 1. 19. An expressionvector comprising an expression cassette operably linked to the nucleicacid molecule of claim
 18. 20. The expression vector of claim 19,wherein the expression vector is an M13 phage-based plasmid.
 21. A hostcell comprising the vector of claim
 19. 22. A method of preparing afibronectin type III (Fn3) polypeptide monobody comprising the steps of:a) providing a DNA sequence encoding a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein at least one loop region contains a unique restriction enzymesite, and wherein at least one of the plurality of Fn3 β-strand domainsequences are more stable at neutral pH than wild-type Fn3; b) cleavingthe DNA sequence at the unique restriction site; c) inserting into therestriction site a DNA segment known to encode a peptide capable ofbinding to a specific binding partner (SBP) or a transition state analogcompound (TSAC) so as to yield a DNA molecule comprising the insertionand the DNA sequence of (a); and d) expressing the DNA molecule so as toyield polypeptide monobody.
 23. A method of preparing a fibronectin typeIII (Fn3) polypeptide monobody comprising the steps of: (a) providing areplicatable DNA sequence encoding a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein the nucleotide sequence of at least one loop region is known,and wherein at least one of the plurality of Fn3 β-strand domainsequences are more stable at neutral pH than wild-type Fn3; (b)preparing polymerase chain reaction (PCR) primers sufficientlycomplementary to the known loop sequence so as to be hybridizable underPCR conditions, wherein at least one of the primers contains a modifiednucleic acid sequence to be inserted into the DNA; (c) performingpolymerase chain reaction using the DNA sequence of (a) and the primersof (b); (d) annealing and extending the reaction products of (c) so asto yield a DNA product; and (e) expressing the polypeptide monobodyencoded by the DNA product of (d).
 24. A method of preparing afibronectin type III (Fn3) polypeptide monobody comprising the steps of:a) providing a replicatable DNA sequence encoding a plurality of Fn3β-strand domain sequences that are linked to a plurality of loop regionsequences, wherein the nucleotide sequence of at least one loop regionis known, and wherein at least one of the plurality of Fn3 β-stranddomain sequences are more stable at neutral pH than wild-type Fn3; b)performing site-directed mutagenesis of at least one loop region so asto create a DNA sequence comprising an insertion mutation; and c)expressing the polypeptide monobody encoded by the DNA sequencecomprising the insertion mutation.
 25. A kit for performing the methodof any one of claims 22-24, comprising a replicatable DNA encoding aplurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein at least one of theplurality of Fn3 β-strand domain sequences are more stable at neutral pHthan wild-type Fn3.
 26. A variegated nucleic acid library encoding Fn3polypeptide monobodies comprising a plurality of nucleic acid specieseach comprising a plurality of loop regions, wherein the species encodea plurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein one or more of the loopregion sequences vary by deletion, insertion or replacement of at leasttwo amino acids from corresponding loop region sequences in wild-typeFn3; wherein the β-strand domain sequences of the monobody have at leasta 50% total amino acid sequence homology to the corresponding amino acidsequences of β-strand domain sequences of the wild-type Fn3; and whereinthe Fn3 is more stable at neutral pH than wild-type Fn
 27. Thevariegated nucleic acid library of claim 26, wherein one or more of theloop regions encodes: i) an AB amino acid loop from residue 15 to 16inclusive; ii) a BC amino acid loop from residue 22 to 30 inclusive;iii) a CD amino acid loop from residue 39 to 45 inclusive; iv) a DEamino acid loop from residue 51 to 55 inclusive; v) an EF amino acidloop from residue 60 to 66 inclusive; and vi) an FG amino acid loop fromresidue 76 to 87 inclusive.
 28. The variegated nucleic acid library ofclaim 26, wherein the loop region sequences vary from the wild-type Fn3loop region sequences by the deletion or replacement of at least 2 aminoacids.
 29. The variegated nucleic acid library of claim 26, wherein themonobody loop region sequences vary from the wild-type Fn3 loop regionsequences by the insertion of from 3 to 25 amino acids.
 30. Thevariegated nucleic acid library of claim 26, wherein a variegatednucleic acid sequence comprising from 6 to 75 nucleic acid bases isinserted in any one of the loop regions of the species.
 31. Thevariegated nucleic acid library of claim 26, wherein the variegatedsequence is constructed so as to avoid one or more codons selected fromthe group consisting of those codons encoding cysteine or the stopcodon.
 32. The variegated nucleic acid library of claim 26, wherein thevariegated nucleic acid sequence is located in the BC loop.
 33. Thevariegated nucleic acid library of claim 26, wherein the variegatednucleic acid sequence is located in the DE loop.
 34. The variegatednucleic acid library of claim 26, wherein the variegated nucleic acidsequence is located in the FG loop.
 35. The variegated nucleic acidlibrary of claim 26, wherein the variegated nucleic acid sequence islocated in the AB loop.
 36. The variegated nucleic acid library of claim26, wherein the variegated nucleic acid sequence is located in the CDloop.
 37. The variegated nucleic acid library of claim 26, wherein thevariegated nucleic acid sequence is located in the EF loop.
 38. Apeptide display library derived from the variegated nucleic acid libraryof claim
 26. 39. A peptide display library of claim 38, wherein thepeptide is displayed on the surface of a bacteriophage or virus.
 40. Apeptide display library of claim 39, wherein the bacteriophage is M13 orfd.
 41. A method of identifying the amino acid sequence of a polypeptidemolecule capable of binding to a specific binding partner (SBP) so as toform a polypeptide:SSP complex wherein the dissociation constant of thepolypeptide:SBP complex is less than 10⁻⁶ moles/liter, comprising thesteps of: a) providing a peptide display library according to claim 39;b) contacting the peptide display library of (a) with an immobilized orseparable SBP; c) separating the peptide:SBP complexes from the freepeptides, d) causing the replication of the separated peptides of (c) soas to result in a new peptide display library distinguished from that in(a) by having a lowered diversity and by being enriched in displayedpeptides capable of binding the SBP; e) optionally repeating steps (b),(c), and (d) with the new library of (d); and f) determining the nucleicacid sequence of the region encoding the displayed peptide of a speciesfrom (d) and deducing the peptide sequence capable of binding to theSBP.
 42. A method of preparing a variegated nucleic acid libraryencoding Fn3 polypeptide monobodies having a plurality of nucleic acidspecies each comprising a plurality of loop regions, wherein the speciesencode a plurality of Fn3β-strand domain sequences that are linked to aplurality of loop region sequences, wherein one or more of the loopregion sequences vary by deletion, insertion or replacement of at leasttwo amino acids from corresponding loop region sequences in wild-typeFn3, and wherein the β-strand domain sequences of the monobody have atleast a 50% total amino acid sequence homology to the correspondingamino acid sequences of β-strand domain sequences of the wild-type Fn3,and wherein the Fn3 comprises a stabilizing mutation β-strand domain,comprising the steps of a) preparing an Fn3 polypeptide monobody havinga predetermined sequence; b) contacting the polypeptide with a specificbinding partner (SBP) so as to form a polypeptide:SSP complex whereinthe dissociation constant of the polypeptide:SBP complex is less than10⁻⁶ moles/liter; c) determining the binding structure of thepolypeptide:SBP complex by nuclear magnetic resonance spectroscopy orX-ray crystallography; and d) preparing the variegated nucleic acidlibrary, wherein the variegation is performed at positions in thenucleic acid sequence which, from the information provided in (c),result in one or more polypeptides with improved binding to the SBP. 43.A method of identifying the amino acid sequence of a polypeptidemolecule capable of catalyzing a chemical reaction with a catalyzed rateconstant, k_(cat), and an uncatalyzed rate constant, L_(uncat), suchthat the ratio of k_(cat)/k_(uncat) is greater than 10, comprising thesteps of: a) providing a peptide display library according to claim 39;b) contacting the peptide display library of (a) with an immobilized orseparable transition state analog compound (TSAC) representing theapproximate molecular transition state of the chemical reaction; c)separating the peptide:TSAC complexes from the free peptides; d) causingthe replication of the separated peptides of (c) so as to result in anew peptide display library distinguished from that in (a) by having alowered diversity and by being enriched in displayed peptides capable ofbinding the TSAC; e) optionally repeating steps (b), (c), and (d) withthe new library of (d); and f) determining the nucleic acid sequence ofthe region encoding the displayed peptide of a species from (d) andhence deducing the peptide sequence.
 44. A method of preparing avariegated nucleic acid library encoding Fn3 polypeptide monobodieshaving a plurality of nucleic acid species each comprising a pluralityof loop regions, wherein the species encode a plurality of Fn3 β-stranddomain sequences that are linked to a plurality of loop regionsequences, wherein one or more of the loop region sequences vary bydeletion, insertion or replacement of at least two amino acids fromcorresponding loop region sequences in wild-type Fn3, and wherein theβ-strand domain sequences of the monobody have at least a 50% totalamino acid sequence homology to the corresponding amino acid sequencesof β-strand domain sequences of the wild-type Fn3, and wherein the Fn3comprises a stabilizing mutation β-strand domain, comprising the stepsof a) preparing an Fn3 polypeptide monobody having a predeterminedsequence, wherein the polypeptide is capable of catalyzing a chemicalreaction with a catalyzed rate constant, k_(cat), and an uncatalyzedrate constant, k_(uncat), such that the ratio of k_(cat)/k_(uncat) isgreater than 10; b) contacting the polypeptide with an immobilized orseparable transition state analog compound (TSAC) representing theapproximate molecular transition state of the chemical reaction; c)determining the binding structure of the polypeptide:TSAC complex bynuclear magnetic resonance spectroscopy or X-ray crystallography; and d)preparing the variegated nucleic acid library, wherein the variegationis performed at positions in the nucleic acid sequence which, from theinformation provided in (c), result in one or more polypeptides withimproved binding to or stabilization of the TSAC.
 45. An isolatedpolypeptide identified by the method of claim
 41. 46. An isolatedpolypeptide identified by the method of claim
 43. 47. A kit foridentifying the amino acid sequence of a polypeptide molecule capable ofbinding to a specific binding partner (SBP) so as to form apolypeptide:SSP complex wherein the dissociation constant of thepolypeptide:SBP complex is less than 10⁻⁶ moles/liter, comprising thepeptide display library of claim
 39. 48. A kit for identifying the aminoacid sequence of a polypeptide molecule capable of catalyzing a chemicalreaction with a catalyzed rate constant, k_(cat), and an uncatalyzedrate constant, k_(uncat), such that the ratio of k_(cat)/k_(uncat) isgreater than 10, comprising the peptide display library of claim
 39. 49.A polypeptide derived by using the kit of claim
 47. 50. A polypeptidederived by using the kit of claim 48.